Calloway was born in Rochester, New York, and grew up in Baltimore, Maryland, where he sang in the church choir. He dropped out of law school to take a part in a touring show called Plantation Days (1927), and appeared on the Broadway stage in Blackbirds of 1928, singing ‘I Can't Give You Anything But Love’. His first band was the Alabamians (1929-30), followed by his own backing band, which became the most highly paid of the black orchestras in the racially segregated 1930s-40s. Many outstanding jazz musicians played in the Calloway band at one time, including saxophonists Ben Webster (1909-1973) and Chu Berry (1908-41), and trumpeter Dizzy Gillespie.

A handsome and flamboyant figure, Calloway was said to have invented the baggy zoot suit; he was the model for the character Sportin' Life in George Gershwin's opera Porgy and Bess (1935), acting the part in the 1950s.

He appeared in many films, including The Big Broadcast (1932), Manhattan Merry-Go-Round (1937) (both with his orchestra), and Stormy Weather (1943). He starred in the musicals The Pyjama Game (on Broadway) and Hello, Dolly (an all-black version), and also featured in the film The Blues Brothers (1980).