His training began in Canada on piano and organ and continued in the United States and France. He spent most of his adult career in the US as a piano teacher and choir director, the longest period at the Hampton Institute, Hampton, Va. As president (1924-26) and until his death a leading figure of the National Association of Negro Musicians, he advanced the cause of black musicians in general. In particular he espoused the preservation and dissemination of spirituals as compiler, arranger and conductor. With the Hampton Institute's Choral Union he toured Europe in 1930, introducing audiences to black American music.

His compositions include a number of suites for piano which blend elements of blues, salon and romantic concert music in short movements with descriptive titles. His harmonic vocabulary is inventive and often original. The choral-orchestral work The Ordering of Moses earned Dett a master's degree in music from the Eastman School of Music in 1932. A symphony was left incomplete at his death. His "Juba Dance" from the piano suite "In the Bottoms" is one of the most popular pieces by any Canadian composer. It was a favourite piece of English composer Percy Grainger and was also performed by Sousa's band.

In 2002 the first recording of selected choral works by Dett, entitled Listen to the Lambs, was released by the R. Nathaniel Dett Chorale.