He was born in Ferrara, and spent his early life both in Padua and Ferrara, entering the service of Alfonso II d'Este, Duke of Ferrara, in 1567. After 15 years in the service of the Duke, he resigned and withdrew to his family estate, the Villa Guarina, where he wrote his most notable work, Il pastor fido (1590; The Faithful Shepherd, 1647). This play, a pastoral tragicomedy, polished in style, was translated into many languages and became popular during the 17th century. It set the pattern for a code of refinement and gallantry that lasted until the late 18th century.

He was the father of Anna Guarini, one of the famous virtuose singers of the Ferrara court, the three women of the concerto di donne. She was murdered by her husband in 1598, with the assistance of her brother Girolamo.

No poet was more influential on music history in the late Renaissance and Baroque eras than Guarini. His poems were set more often by madrigal composers than the work of any other poet, even Tasso, who came in a close second; the prolific madrigal composer Philippe de Monte even named one of his collections Il pastor fido after Guarini's most famous work. His popularity was due to his providing texts to composers which were rich with possibilities for word - painting and other easy translations of emotion into music. One of his poems, the erotic Tirsi morir volea, recounting the amorous encounter of a shepherd and a nymph, was set to music as a madrigal more often than any other single poem of the entire era. Another of Guarini's poems which was set by numerous madrigalists was Cor mio, deh non languire ("Dear heart, I prithee do not waste away").

In addition to his decisive influence on madrigal composers, he was the single largest influence on opera librettists up until the time of Metastasio in the 18th century.

While Guarini's work may be seen as lacking the deep feeling and sentiment of another poet at the Estensi court, Torquato Tasso, it was precisely this quality which commended it to musical setting at a time when excessive emotionalism had become unfashionable.

An example of his work would be "O come è gran martire" from Libro Terzo dei Madrigali (1592) by Monteverdi.