There is some evidence that he may have spent some time in Verona in the early 1550s, due to a connection with Vincenzo Ruffo, who worked there as maestro di cappella – Ruffo published one of Gabrieli's madrigals in 1554, and Gabrieli also wrote some music for a Veronese academy. Gabrieli is known to have been organist in Cannaregio between 1555 and 1557, at which time he competed unsuccessfully for the post of organist at St. Mark's.

In 1562 he went to Germany, where he visited Frankfurt am Main and Munich; while there he met and became friends with Orlande de Lassus, one of the most wide-ranging composers of the entire Renaissance, who wrote secular songs in French, Italian, and German, as well as abundant Latin sacred music. This musical relationship proved immensely fruitful for both composers: while Lassus certainly learned from the Venetian, Gabrieli took back to Venice numerous ideas he learned while visiting Lassus in Bavaria, and within a short time was composing in most of the current idioms, including one which Lassus entirely avoided: purely instrumental music.

In 1566 Gabrieli was chosen for the post of organist at St. Mark's, one of the most prestigious musical posts in northern Italy; he retained this position for the rest of his life. Around this time he acquired, and maintained, a reputation as one of the finest current composers. Working in the unique acoustical space of St. Mark's, he was able to develop his unique, grand ceremonial style, which was enormously influential in the development of the polychoral style and the concertato idiom, which partially defined the beginning of the Baroque era in music.

His duties at St. Mark's clearly included composition, for he wrote a great deal of music for ceremonial affairs, some of considerable historical interest. He provided the music for the festivities accompanying the celebration of the victory over the Turks in the Battle of Lepanto (1571); he also composed music for the visit of several princes from Japan (1585).

Late in his career he also became famous as a teacher. Prominent among his students were his nephew Giovanni Gabrieli; the music theorist Lodovico Zacconi; Hans Leo Hassler, who carried the concertato style to Germany; and many others.