Doboius was born in Rosnay in Marne. He studied first under Louis Fanart (the choirmaster at Reims cathedral) and later at the Paris Conservatoire. He won the Prix de Rome in 1861. In 1868, he became choirmaster at the Church of the Madeleine, and in 1871 took over from César Franck as organist at the Church of Sainte-Clotilde. In 1877, Dubois returned to the Church of the Madeleine, succeeding Camille Saint-Saëns as organist there. From 1871 he taught at the Paris Conservatoire, with Paul Dukas among his pupils.
Dubois was director of the Conservatoire from 1896 to 1905. He was forced to resign after Romain Rolland wrote a letter criticising him for refusing to award the Prix de Rome to Maurice Ravel. He was succeeded in the post by Gabriel Fauré.
Dubois wrote a variety of music, including operas, oratorios and three symphonies. His best known work is the oratorio Les sept paroles du Christ (1867), although none of his works are regularly performed today. He has had a more lasting influence in teaching, with his theoretical works Traité de contrepoint et de fugue (on counterpoint and fugue) and Traité d'harmonie théorique et pratique (on harmony) still being used today.