He studied at Montpellier with the famous medical professor Guillaume Rondelet, though he never practiced medicine. In 1573 he was appointed prefect of the imperial medical garden in Vienna by Maximilian II and made Gentleman of the Imperial Chamber, but he was discharged from the imperial court shortly after the accession of Rudolf II in 1576. After leaving Vienna in the late 1580s he established himself in Frankfurt am Main, before his appointment as professor at the University of Leiden in October 1593. He helped create one of the earliest formal botanical gardens of Europe at Leyden, the Hortus Academicus, and his detailed planting lists have made it possible to recreate his garden near where it originally lay.

In the history of gardening he is remembered not only for his scholarship but also for his observations on tulips "breaking" — a phenomenon discovered in the late 19th century to be due to a virus — causing the many different flamed and feathered varieties, which led to the speculative tulip mania of the 1630s. Clusius laid the foundations of Dutch tulip breeding and the bulb industry today.

His first publication was a French translation of Rembert Dodoens's herbal, published in Antwerp in 1557 by van der Loë. His Antidotarium sive de exacta componendorum miscendorumque medicamentorum ratione ll. III ... nunc ex Ital. sermone Latini facti (1561) initiated his fruitful collaboration with the renowned Plantin printing press at Antwerp, which permitted him to issue late breaking discoveries in natural history and to ornament his texts with elaborate engravings. Clusius, as he was known to his contemporaries, published two major original works: his Rariorum aliquot stirpium per Hispanias observatarum historia (1576) — is one of the earliest books on Spanish flora — and his Rariorum stirpium per Pannonias observatorum Historiae (1583) is the first book on Austrian and Hungarian alpine flora. His collected works were published in two parts: Rariorum plantarum historia (1601) contains his Spanish and Austrian flora and adds more information about new plants as well as a pioneering mycological study on mushrooms from Central Europe; and Exoticorum libri decem (1605) is an important survey of exotic flora and fauna, both still often consulted. He contributed as well to Abraham Ortelius's map of Spain. Clusius translated several contemporary works in natural science.

Clusius was also among the first to study the flora of Austria, under the auspices of Emperor Maximilian II. He was the first botanist to climb the Ötscher and the Schneeberg in Lower Austria, which was also the first documented ascent of the latter. His contribution to the study of alpine plants has led to many of them being named in his honour, such as Gentiana clusii, Potentilla clusiana and Primula clusiana. The genus Clusia (whence the family Clusiaceae) also honours Clusius.