The earliest beginnings of the gardens go back to 1779, when the Accademia dei Regi Studi created the chair of "Botany and medicinal properties". A modest plot of land was allocated to develop a small botanical garden dedicated to the cultivation of plants with medicinal benefits, for the twin objectives of general learning and improving public health.

This initial garden allotment soon proved insufficient for the purposes for which it was intended, and in 1786 it was decided to move to the present site, right next to the Piano di Sant'Erasmo, best remembered for the unfortunate events that occurred there during the Spanish Inquisition. In 1789 construction of the main part of the administrative buildings of the garden commenced in aneoclassical style. It is constructed with a central building, the Gymnasium, and two side buildings, the Tepidariumand the Caldarium, designed by the Frenchman Léon Dufourny, who had also designed a part of the oldest section of garden, right next to the Gymnasium. Its rectangular layout is divided into four quadrangles, within which the species are categorised according to Carolus Linnaeus' system of classification. The new garden was opened in 1795; in the ensuing years it was improved, with the Aquarium (1798), a great pool hosting numerous species of aquatic plants, and the serra Maria Carolina (or Maria Carolina glasshouse), completed in 1823. The huge Ficus macrophylla, which is an emblem and a well-known attraction of the modern garden, was imported from Norfolk Island (Australia), in 1845. Today's area, some 10 hectares, was reached in 1892, following successive extensions. In 1913 the Giardino coloniale (Colonial garden) was developed alongside the botanic gardens, but that no longer exists. The gardens have been managed by the Department of Botany since 1985.