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.

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.

That year Spon published his Histoire de la république de Genève, followed by his Récherches curieuses d'antiquité (Lyon 1683) and in 1685 a collection of transcriptions of Roman inscriptions gleaned over the years, Miscellanea eruditae antiquitatis, in the preface to which he offered one of the earliest definitions of "archaeologia" to describe the study of antiquities in which he was engaged.

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.