William Clarence Eckstine was born on July 8, 1914, in Pittsburgh, Pennsylvania, the son of William Eckstein, a chauffeur, and Charlotte Eckstein, a seamstress. His sister Maxine became a high school teacher. He attended Peabody High School in Pittsburgh, then moved to Washington, D.C., where he went to Armstrong High School, St. Paul Normal and Industrial School and Howard University. In 1933, at nineteen, he left Howard to start a music career after winning first prize, $10, in an amateur contest at Washington's Howard Theatre with an imitation of Cab Calloway singing a nursery rhyme with scat syllables added.

He went to Chicago and in 1939 joined Earl Hines's Grand Terrace Orchestra as vocalist and trumpeter, staying until 1943. With Hines he had juke-box hits in "Jelly, Jelly," which he co-wrote, and "Stormy Monday Blues," recorded in 1942, which topped the Harlem Hit Parade. He and the tenor saxophonist and arranger Budd Johnson are credited with helping steer Hines toward younger players, and the band Eckstine left in late 1943 had carried Dizzy Gillespie, Charlie Parker and the singer Sarah Vaughan, whom he is credited with recommending to Hines after hearing her at the Apollo Theater.

In 1944 he formed his own big band, generally considered the first bebop big band: a working orchestra in which the new harmonic and rhythmic language of Gillespie and Parker was played night after night. Over its life the band carried Parker, Gillespie, Dexter Gordon, Gene Ammons, Art Blakey, Cecil Payne, Fats Navarro, Lucky Thompson and John Malachi, along with Miles Davis for stretches, with Tadd Dameron, Gil Fuller and Jerry Valentine among its arrangers. Ammons joined in 1944, and Eckstine gave him the nickname "Jug" when straw hats ordered for the band did not fit him. Vaughan joined in 1944 and left to go out on her own before the year was out. A DeLuxe session on December 5, 1944 produced "Blowin' the Blues Away," on which Gordon and Ammons trade choruses, Eckstine's own ballad "I Want to Talk About You," and Vaughan's first record, "I'll Wait and Pray." Gillespie left the band in 1944 to lead small groups and recommended Navarro in his place. Davis sat in during the band's July 1944 engagement in St. Louis and was a member in 1946, recording with it in Los Angeles that October alongside Payne, and Lena Horne sang with the orchestra in 1945 and 1946. Blakey held the drum chair from 1944 to 1947.

The orchestra's records for DeLuxe and then National sold well as well as breaking ground. Eckstine's 1945 versions of "A Cottage for Sale," which reached number eight on the pop chart, and "Prisoner of Love" each earned a gold disc from the RIAA. Eckstine starred as the hero of the musical film Rhythm in a Riff, made with his orchestra. He disbanded the orchestra in 1947 and became a solo singer.

The solo career was the larger commercial one. Eckstine signed with the newly founded MGM label and had hits with revivals of "Everything I Have Is Yours" (1947), "Blue Moon" (1948) and Juan Tizol's "Caravan" (1949), then "My Foolish Heart" in 1950 and "I Apologize" in 1951, the last of which was given a Grammy Hall of Fame Award in 1999. A three-page profile in Life on April 24, 1950 included a photograph by Martha Holmes of Eckstine surrounded by white female admirers; the magazine's publisher approved it for print, protest letters followed, and his biographer Cary Ginell and the pianist Billy Taylor are among those who say the episode cost him part of his white pop audience. The press called him "the sepia Sinatra," and the New York Times reported that his 1950 booking at New York's Paramount Theatre drew a larger crowd than Frank Sinatra's had. In the mid-1950s he recorded for MGM, EmArcy and Mercury, among them a set of Duke Ellington songs and the album Blues for Sale, and in 1957 he and Vaughan made the duet single "Passing Strangers," which reached number 22 in the UK, together with the album Sing the Best of Irving Berlin.

His later jazz-leaning dates were often made with colleagues from the orchestra years. Basie/Eckstine Incorporated (Roulette, 1959) paired him with the Count Basie Orchestra and was the only recorded collaboration between the two, with arrangements by Quincy Jones, Thad Jones and Bobby Tucker. His 1960 Las Vegas set No Cover, No Minimum included a few trumpet solos, and in 1962 he recorded At Basin Street East with Quincy Jones. He then recorded for Motown in the mid and late 1960s and, sparingly, for the Stax-owned Enterprise label in the 1970s. He sang on television variety programs, appeared in films and in an episode of the sitcom Sanford and Son, and sang the national anthem before Game 4 of the 1979 World Series in his native Pittsburgh.

In 1984 he made I Am a Singer, arranged and conducted by Angelo DiPippo with Toots Thielemans on harmonica. In November 1986 he recorded his last album, Billy Eckstine Sings with Benny Carter, with the saxophonist Benny Carter and the singer Helen Merrill as a guest; it was nominated for a Grammy Award for best male jazz vocal performance. He married June in 1942 and, after their divorce in 1952, the actress and model Carolle Drake in 1953, and the second marriage lasted until his death. Of their four children, Ed Eckstine became president of Mercury Records and Guy Eckstine a record producer and A&R executive. Eckstine suffered a stroke while performing in Salina, Kansas, in April 1992 and never performed again. He had a heart attack and passed away in Pittsburgh on March 8, 1993, at age 78; his last word is reported to have been "Basie." A state historical marker stands at 5913 Bryant Street in Pittsburgh's Highland Park, where he grew up, and in 2019 he was posthumously given the Grammy Lifetime Achievement Award.

Eckstine sang in a rich, almost operatic bass-baritone, and the New York Times credited his full-throated, sugary approach to popular songs with shaping singers such as Earl Coleman, Johnny Hartman, Joe Williams, Arthur Prysock and Lou Rawls. He was also a working instrumentalist: he played trumpet in the Hines band, took valve trombone solos on his orchestra's April 1947 Los Angeles session and on a Chicago small-group date led by Howard McGhee, and played a few trumpet solos on his 1960 live album. Duke Ellington, Quincy Jones and Lionel Hampton all paid public tribute to him, Jones calling him an idol whose dress, speech and bearing he wanted to copy, and his high-rolled "Mr. B. Collar" was widely imitated by hipsters in the late 1940s and early 1950s. Gillespie recalled in his autobiography that no band sounded like Eckstine's, with its strong attack and modern style. The jazz weight of his catalogue sits in the 1944 to 1947 orchestra and in later dates with Basie, Vaughan and Benny Carter, while many of the MGM, Motown and Stax records are orchestral pop balladry.