Born into a lineage of New Orleans musicians as the grandson of trumpeter and music teacher Jim Humphrey, the son of clarinetist Willie Humphrey, Sr., and the brother of trumpeter Percy Humphrey and trombonist Earl Humphrey, Willie Humphrey, Jr. followed the family path into music. After initial violin studies, he took up the clarinet at age 14 and began performing in local settings. During 1919-1920 he lived briefly in Chicago, appearing with King Oliver and Freddie Keppard, yet soon returned home and thereby bypassed an early chance at recording. Between 1925 and 1932 he resided in St. Louis, working with Fate Marable and Dewey Jackson, and he later toured with Lucky Millinder from 1935 to 1936; otherwise he remained in New Orleans for the rest of his life. During World War II he taught music and played in a Navy band, while the 1950s found him performing for a time with Paul Barbarin. Humphrey and his brother Percy achieved widespread recognition beginning in the 1960s as members of the Preservation Hall Jazz Band, carrying New Orleans jazz to audiences worldwide through extensive touring. Although never a virtuoso, he delivered his straightforward, ensemble-oriented style with genuine spirit. As a leader he recorded for Smoky Mary and, late in his career, for GHB.
