Shirley Valerie Horn was born on May 1, 1934, in Washington, D.C. Her grandmother, an amateur organist, pushed her toward music, and she began piano lessons at four. As a teenager she studied classical piano and composition at Howard University, and she was offered a place at the Juilliard School that her family could not afford to let her take. Her models at the keyboard were Erroll Garner, Oscar Peterson and Ahmad Jamal; she later said Peterson became her Rachmaninoff and Jamal her Debussy. She formed her first jazz trio at twenty, in 1954, slipped into the clubs along Washington's U Street before she was old enough to be admitted, and in 1955 married Sheppard Deering, with whom she had a daughter, Rainy.

Her recording career began in 1959, when she played in one of the rhythm sections on the violinist Stuff Smith's Verve album Cat on a Hot Fiddle, though the original LP left her name off the list of musicians. In 1960 the small New York label Stere-O-Craft recorded her debut as a leader, Embers and Ashes, a vocal-and-piano set with bass and drums. A copy reached Miles Davis, who praised it publicly, which he rarely did for other musicians, and who invited her to play the intermission sets during his dates at the Village Vanguard in 1961. A live set she recorded in 1961 at St. Louis's Gaslight Square district was later issued as "Live" at the Village Vanguard and reissued on CD as At the Gaslight Square 1961.

Davis's backing led to a Mercury contract. In 1962 Quincy Jones, then a Mercury executive, signed her, and the label gave her two orchestral LPs in 1963, Loads of Love, arranged by Jimmy Jones with Hank Jones on piano and a band that included Kenny Burrell, Gerry Mulligan and Al Cohn, and Shirley Horn with Horns, with arrangements by Quincy Jones, Thad Jones, Billy Byers and Don Sebesky. A third Mercury album was recorded and never released. She made Travelin' Light for ABC-Paramount in 1965, covering the Beatles' "And I Love Him," but she mostly resisted efforts to remake her as a pop singer, and she sang the end-title song for Quincy Jones's score to the film For Love of Ivy (1968). From the late 1960s to the late 1970s she worked mostly around Washington, raising her daughter, and she made one album in those years, Where Are You Going, on Perception Records in 1972, and did not tour to promote it.

In 1978 the Danish label SteepleChase tracked her down in Washington and offered a trio session with the drummer Billy Hart, whom she had known for years, and the bassist Buster Williams. A Lazy Afternoon was the first of four SteepleChase albums recorded between 1978 and 1984. Hart's own profile says she hired him out of a Washington club while he was a Howard University student and took him on the road. Playing the North Sea Jazz Festival in The Hague in July 1981, where two of her albums were taped, brought her back before international audiences, and she returned to New York clubs in 1982. In 1986 she made a one-off record for CBS-Sony in Japan, All of Me, with Frank Wess as a guest on three tracks, and in May 1987 she recorded the live I Thought About You in Hollywood, her first album for Verve. After a stop at Audiophile Records for Softly, she settled at Verve, for which she made eleven studio and live albums during her lifetime.

The Verve years were the most successful of her career. Her working trio was Charles Ables on electric bass and Steve Williams on drums, a rhythm section she kept for roughly a quarter of a century. Close Enough for Love (1989) featured the Washington tenor saxophonist Buck Hill on five tracks. You Won't Forget Me (1991) was her first number one on the jazz chart and the last album on which Miles Davis appeared as a sideman, playing on the title song; Wynton Marsalis, Branford Marsalis, Toots Thielemans and Buck Hill were also among the guests. Here's to Life (1992), with orchestral arrangements by Johnny Mandel, produced the title song that became her signature and won Mandel a Grammy Award for his arrangements. Light Out of Darkness (A Tribute to Ray Charles) (1993) and I Love You, Paris, taped in March 1992 at the Théâtre du Châtelet, also reached number one on the Billboard jazz chart. She won the Grammy Award for Best Jazz Vocal Performance at the 41st ceremony for I Remember Miles (1998), her tribute to Davis, whose own drawing of the two of them was used on the cover. She was nominated for nine Grammys in all.

Illness slowed her in the early 2000s. She was a breast cancer survivor, and complications of diabetes led to the amputation of her right foot, after which she sang seated and handed the piano to her accompanist George Mesterhazy; he played most of the piano on her last studio album, May the Music Never End (2003), with Ahmad Jamal as guest on two tracks. She returned to playing piano in public in 2004 and made her final recordings in January 2005 at a New York club. The National Endowment for the Arts named her a Jazz Master in 2005; she had received an honorary doctorate from Berklee in 2002, and the 109th Congress formally recognized her contributions. She passed away on October 20, 2005, at age 71, from complications of diabetes, at a nursing home in Cheverly, Maryland, and was buried at Fort Lincoln Cemetery near Washington. Concert recordings from her last decades, including Live at the 1994 Monterey Jazz Festival (2008) and Live at the 4 Queens (2016), a 1988 Las Vegas set, have appeared since.

Horn's signature was tempo. She sang ballads slower than almost any of her contemporaries, holding notes, leaving silences, stressing some words and letting others fade, and her voice was a smoky contralto. She accompanied herself on a spare piano with unusual independence between hands and voice; the arranger Johnny Mandel likened her ability to play and sing independently at once to having two heads, and he compared her piano skill with Bill Evans's. She turned from her classical training to the jazz pianists Erroll Garner, Oscar Peterson and Ahmad Jamal, and Miles Davis, who rarely praised other musicians in public, was her most important early champion. Her working rhythm section was part of the sound: writing in the Los Angeles Times in 1995, Don Heckman credited bassist Charles Ables and drummer Steve Williams with following her every spontaneous turn. Her Mercury albums put her in conventional orchestral settings, and a reviewer of those records noted that she was persuaded to sing faster there than she later would, a contrast with her later trio records. She recorded standards and ballads along with some pop and soul material, including the Beatles' "And I Love Him" and a full album of Ray Charles songs, and she remained a jazz singer and pianist throughout. Younger musicians have cited her phrasing: the trumpeter Josh Evans names her elegant, unhurried phrasing as an influence on his melodic approach, and the Italian pianist-singer Silvia Manco places her own piano-and-vocal trio in a line of singer-pianists that includes Horn.