Tournefort was born in Aix-en-Provence and studied there in the convent of the Jesuits. He was destined for the Church, but the death of his father left him free to follow his botanical inclinations. After two years collecting, he studied medicine at Montpellier, but was appointed professor of botany at the Jardin des Plantes in 1683. By the king's order he travelled through western Europe, particularly the Pyrenees, where he made extensive collections. Between 1700 and 1702 Tournefort travelled through the islands of Greece and visited Constantinople, the borders of the Black Sea, Armenia, and Georgia, collecting plants and undertaking other types of observations. He was accompanied by the German botanist Andreas Gundesheimer (1668-1715) and the artist Claude Aubriet (1651-1743). His description of this journey was published posthumously (Relation d'un voyage du Levant).
His principal work was Institutiones rei herbariae (1700), which classified plants according to the form of the corolla, but more importantly made a clear distinction between genus and species, and as such prepared the way for Linnaeus. He was run over and killed by a carriage in Paris in the road which now bears his name (Rue de Tournefort in the 5ème arrondissement).
His other works included De optima methode instituenda in re herbaria (1697), and Histoire des plantes qui naissent aux environs de Paris (1698), an English translation of which appeared in 1732. A genus belonging to the family of the Boraginaceae was named by Linnaeus Tournefortia in his honour.