Giovanni studied with his uncle, the composer Andrea Gabrieli, and with Orlande de Lassus. He became principal organist at the church of San Marco in Venice in 1585, and following his uncle's death the following year also took the post of principal composer. San Marco had a long tradition of musical excellence and Gabrieli's work there made him one of the most noted composers in Europe. Like composers before and after him, he would use the unusual layout of the church, with its two choir lofts facing each other, to create striking spatial effects. Several of his pieces are written so that a choir will first be heard from the left, followed by a response from the choir to the right.
Gabrieli was quite an original composer, and is seen by many as an important transitional figure between the renaissance and baroque periods. In his works can be found early uses of basso continuo and, in the Sonata pian e forte, some of the earliest dynamic markings (about how loud or quiet the music should be). He was also one of the first to use instrumental parts in choral works. In 1597 he published the collection of vocal music Sacrae symphoniae which become known across the continent. He later taught figures such as Michael Praetorius and Heinrich Schutz.