Gabrieli was born in Venice. He was one of five children, and his father came from the region of Carnia and went to Venice shortly before Giovanni's birth. While not much is known about Giovanni's early life, he probably studied with his uncle, the composer Andrea Gabrieli; he may indeed have been brought up by him, as is implied by the dedication to his 1587 book of concerti, in which he described himself as "little less than a son" to his uncle. He also went to Munich to study with the renowned Orlando de Lassus at the court of Duke Albert V; most likely he stayed there until about 1579. Lassus was to be one of the principal influences on the development of his musical style.
By 1584 he had returned to Venice, where he became principal organist at Saint Mark's Basilica in 1585, after Claudio Merulo left the post; following his uncle's death the following year he took the post of principal composer as well. Also after his uncle's death he began editing much of the older man's music, which would otherwise have been lost; Andrea evidently had had little inclination to publish his own music, but Giovanni's opinion of it was sufficiently high that he devoted much of his own time to compiling and editing it for publication.
Gabrieli's career rose further when he took the additional post of organist at the Scuola Grande di San Rocco, another post he retained for his entire life. San Rocco was the most prestigious and wealthy of all the Venetian confraternities, and second only to San Marco itself in splendor of its musical establishment. Some of the most renowned singers and instrumentalists in Italy performed there and a vivid description of its musical activity survives in the travel memoirs of the English writer Thomas Coryat. Much of his music was written specifically for that location, although he probably composed even more for San Marco.
San Marco had a long tradition of musical excellence and Gabrieli's work there made him one of the most noted composers in Europe. The vogue that began with his influential volume Sacrae symphoniae (1597) was such that composers from all over Europe, especially from Germany, came to Venice to study. Evidently he also made his new pupils study the madrigals being written in Italy, so not only did they carry back the grand Venetian polychoral style to their home countries, but also the more intimate style of madrigals; Heinrich Schütz and others helped transport the transitional early Baroque music north to Germany, a trend that decisively affected subsequent music history. The productions of the German Baroque, culminating in the music of J.S. Bach, were founded on this strong tradition, which had its roots in Venice.
Gabrieli was increasingly ill after about 1606, at which time church authorities began to appoint deputies to take over duties he could no longer perform. He died in 1612, of complications from a kidney stone.