Boyle was born at Lismore Castle, in County Waterford, Ireland, the seventh son and fourteenth child of The 1st Earl of Cork ('the Great Earl of Cork') and Catherine Fenton. Lord Cork, then known simply as Richard Boyle, had arrived in Dublin from England in 1588 during the Tudor plantations of Ireland and obtained an appointment as a deputy escheator. He had amassed enormous wealth and landholdings by the time Robert was born, and had been created Earl of Cork in October 1620. Catherine Fenton, Countess of Cork, was the daughter of Sir Geoffrey Fenton, the former Secretary of State for Ireland, who was born in Dublin in 1539, and Alice Weston, the daughter of Robert Weston, who was born in Lismore in 1541.

Among the critics of the views put forward in this book was a Jesuit, Francis Line (1595–1675), and it was while answering his objections that Boyle made his first mention of the law that the volume of a gas varies inversely to the pressure of the gas, which among English-speaking people is usually called Boyle's Law after his name. The person who originally formulated the hypothesis was Henry Power in 1661. Boyle in 1662 included a reference to a paper written by Power, but mistakenly attributed it to Richard Towneley. In continental Europe the hypothesis is sometimes attributed to Edme Mariotte, although he did not publish it until 1676 and was likely aware of Boyle's work at the time.

As a Director of the East India Company he spent large sums in promoting the spread of Christianity in the East, contributing liberally to missionary societies and to the expenses of translating the Bible or portions of it into various languages. Boyle supported the policy that the Bible should be available in the vernacular language of the people. An Irish language version of the New Testament was published in 1602 but was rare in Boyle's adult life. In 1680–85 Boyle personally financed the printing of the Bible, both Old and New Testaments, in Irish. In this respect, Boyle's attitude to the Irish language differed from the English Ascendancy class in Ireland at the time, which was generally hostile to the language and largely opposed the use of Irish (not only as a language of religious worship).

During this time, his Father hired a private tutor, Robert Carew, who had knowledge of Irish, to act as private tutor to his sons in Eton. However, "only Mr. Robert sometimes desires it [Irish] and is a little entered in it", but despite the "many reasons" given by Carew to turn their attentions to it, "they practice the French and Latin but they affect not the Irish". After spending over three years at Eton, Robert travelled abroad with a French tutor. They visited Italy in 1641 and remained in Florence during the winter of that year studying the "paradoxes of the great star-gazer" Galileo Galilei, who was elderly but still living in 1641.

Robert returned to England from continental Europe in mid-1644 with a keen interest in scientific research. His Father, Lord Cork, had died the previous year and had left him the manor of Stalbridge in Dorset as well as substantial estates in County Limerick in Ireland that he had acquired. Robert then made his residence at Stalbridge House, between 1644 and 1652, and conducted many experiments there. From that time, Robert devoted his life to scientific research and soon took a prominent place in the band of enquirers, known as the "Invisible College", who devoted themselves to the cultivation of the "new philosophy". They met frequently in London, often at Gresham College, and some of the members also had meetings at Oxford.

Having made several visits to his Irish estates beginning in 1647, Robert moved to Ireland in 1652 but became frustrated at his inability to make progress in his chemical work. In one letter, he described Ireland as "a barbarous country where chemical spirits were so misunderstood and chemical instruments so unprocurable that it was hard to have any Hermetic thoughts in it."

In 1654, Boyle left Ireland for Oxford to pursue his work more successfully. An inscription can be found on the wall of University College, Oxford, the High Street at Oxford (now the location of the Shelley Memorial), marking the spot where Cross Hall stood until the early 19th century. It was here that Boyle rented rooms from the wealthy apothecary who owned the Hall.

Reading in 1657 of Otto von Guericke's air pump, he set himself with the assistance of Robert Hooke to devise improvements in its construction, and with the result, the "machina Boyleana" or "Pneumatical Engine", finished in 1659, he began a series of experiments on the properties of air. An account of Boyle's work with the air pump was published in 1660 under the title New Experiments Physico-Mechanical, Touching the Spring of the Air, and its Effects.

In addition to philosophy, Boyle devoted much time to theology, showing a very decided leaning to the practical side and an indifference to controversial polemics. At the Restoration of the king in 1660, he was favourably received at court and in 1665 would have received the provostship of Eton College had he agreed to take holy orders, but this he refused to do on the ground that his writings on religious subjects would have greater weight coming from a layman than a paid minister of the Church.

As a founder of the Royal Society, he was elected a Fellow of the Royal Society (FRS) in 1663. Boyle's law is named in his honour. The Royal Society of Chemistry issues a Robert Boyle Prize for Analytical Science, named in his honour. The Boyle Medal for Scientific Excellence in Ireland, inaugurated in 1899, is awarded jointly by the Royal Dublin Society and The Irish Times. Launched in 2012, The Robert Boyle Summer School organized by the Waterford Institute of Technology with support from Lismore Castle, is held annually to honor the heritage of Robert Boyle.

In 1668 he left Oxford for London where he resided at the house of his elder sister Katherine Jones, Lady Ranelagh, in Pall Mall. His contemporaries widely acknowledged Katherine's influence on his work, but later historiographies dropped her from the record. Theirs was "a lifelong intellectual partnership, where brother and sister shared medical remedies, promoted each other’s scientific ideas, and edited each other’s manuscripts."

In 1669 his health, never very strong, began to fail seriously and he gradually withdrew from his public engagements, ceasing his communications to the Royal Society, and advertising his Desire to be excused from receiving guests, "unless upon occasions very extraordinary", on Tuesday and Friday forenoon, and Wednesday and Saturday afternoon. In the leisure thus gained he wished to "recruit his spirits, range his papers", and prepare some important chemical investigations which he proposed to leave "as a kind of Hermetic legacy to the studious disciples of that art", but of which he did not make known the nature. His health became still worse in 1691, and he died on 31 December that year, just a week after the death of the sister with whom he had lived for more than twenty years. Boyle died from paralysis. He was buried in the churchyard of St Martin-in-the-Fields, his funeral sermon being preached by his friend Bishop Gilbert Burnet. In his will, Boyle endowed a series of Lectures which came to be known as the Boyle Lectures.

Moreover, Boyle incorporated his scientific interests into his theology, believing that natural philosophy could provide powerful evidence for the existence God. In works such as Disquisition about the Final Causes of Natural Things (1688), for instance, he criticised contemporary Philosophers - such as René Descartes - who denied that the study of nature could reveal much about God. Instead, Boyle argued that natural Philosophers could use the design apparently on display in some parts of nature to demonstrate God's involvement with the world. He also attempted to tackle complex theological questions using methods derived from his scientific practices. In Some Physico-Theological Considerations about the Possibility of the Resurrection (1675), he used a chemical experiment known as the reduction to the pristine state as part of an attempt to demonstrate the physical possibility of the resurrection of the body. Throughout his career, Boyle tried to show that science could lend support to Christianity.

Robert Boyle was an alchemist; and believing the transmutation of metals to be a possibility, he carried out experiments in the hope of achieving it; and he was instrumental in obtaining the repeal, in 1689, of the statute of Henry IV against multiplying gold and silver. With all the important work he accomplished in physics – the enunciation of Boyle's law, the discovery of the part taken by air in the propagation of sound, and investigations on the expansive force of freezing water, on specific gravities and refractive powers, on crystals, on electricity, on colour, on hydrostatics, etc. – chemistry was his peculiar and favourite study. His first book on the subject was The Sceptical Chymist, published in 1661, in which he criticised the "experiments whereby vulgar Spagyrists are wont to endeavour to evince their Salt, Sulphur and Mercury to be the true Principles of Things." For him chemistry was the science of the composition of substances, not merely an adjunct to the arts of the Alchemist or the physician. He endorsed the view of elements as the undecomposable constituents of material bodies; and made the distinction between mixtures and compounds. He made considerable progress in the technique of detecting their ingredients, a process which he designated by the term "analysis". He further supposed that the elements were ultimately composed of particles of various sorts and sizes, into which, however, they were not to be resolved in any known way. He studied the chemistry of combustion and of respiration, and conducted experiments in physiology, where, however, he was hampered by the "tenderness of his nature" which kept him from anatomical dissections, especially vivisections, though he knew them to be "most instructing".