His father was Charles Spon, a doctor and Hellenist, of a wealthy and cultured Calvinist banking family from Ulm that had been established since 1551 at Lyon, where they were members of the bourgeois élite.

There his first publication appeared, a Recherche des antiquités et curiosités de la ville de Lyon and he entered into correspondence with a wider circle of savants: the abbé Claude Nicaise at Dijon, du Cange at Paris, the erudite circles that gravitated to le Grand Dauphin and the duc d'Aumont.

Spon traveled to Italy, and then to Greece, to Constantinople and the Levant in 1675–1676 in the company of the English connoisseur and botanist Sir George Wheler (1650–1723), whose collection of antiquities was afterwards bequeathed to Oxford University.

Spon points out that he is an expert on fevers because Lyon includes a swampy area (the Dombes) that produces "mauvais air" responsible for fevers—probably actually malaria.

His money and baggage were stolen from him, and in fragile health, he died of tuberculosis in the canton hospital at Vevey, Christmas Day 1685, at the age of 38.