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, who was employed at St Mark's Basilica from the 1560s until his death in 1585. Giovanni may indeed have been brought up by his uncle, 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.

Giovanni 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 St 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 first motets were published alongside his uncle Andrea's compositions in his 1587 volume of Concerti. These pieces show much influence of his uncle's style in the use of dialogue and echo effects. There are low and high choirs and the difference between their pitches is marked by the use of instrumental accompaniment. The motets published in Giovanni's 1597 Sacrae Symphoniae seem to move away from this technique of close antiphony towards a model in which musical material is not simply echoed, but developed by successive choral entries. Some motets, such as Omnes Gentes developed the model almost to its limits. In these motets, instruments are an integral part of the performance, and only the choirs marked "Capella" are to be performed by Singers for each part.

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.

There seems to be a distinct change in Gabrieli's style after 1605, the year of publication of Monteverdi's Quinto libro di madrigali, and Gabrieli's compositions are in a much more homophonic style as a result. There are sections purely for instruments – called "Sinfonia" – and small sections for soloists singing florid lines, accompanied simply by a basso continuo. "Alleluia" refrains provide refrains within the structure, forming rondo patterns in the motets, with close dialogue between choirs and soloists. In particular, one of his best-known pieces, In Ecclesiis, is a showcase of such polychoral techniques, making use of four separate groups of instrumental and singing performers, underpinned by the omnipresent organ and continuo.

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 in Venice, of complications from a kidney stone.

Also referred to as Symphoniae Sacrae Liber Secundus. Published posthumously in 1615.

Though Gabrieli composed in many of the forms current at the time, he preferred sacred vocal and instrumental music. All of his secular vocal music is relatively early; he never wrote lighter forms, such as dances; and late in his career he concentrated on sacred vocal and instrumental music that exploited sonority for maximum effect. Among the innovations credited to him – and while he was not always the first, he was the most famous to do these things – were the use of dynamics; the use of specifically notated instrumentation (as in the famous Sonata pian' e forte); and the use of massive forces arrayed in multiple, spatially separated groups, an idea which was to be the genesis of the Baroque concertato style, and which spread quickly to northern Europe, both by the report of visitors to Venice and by Gabrieli's students, which included Hans Leo Hassler and Heinrich Schütz.