Tyndall was born in Leighlinbridge, County Carlow, Ireland. His father was a local police constable, descended from Gloucestershire emigrants who settled in southeast Ireland around 1670. Tyndall attended the local schools (Ballinabranna Primary School) in County Carlow until his late teens, and was probably an assistant Teacher near the end of his time there. Subjects learned at school notably included technical drawing and mathematics with some applications of those subjects to land surveying. He was hired as a draftsman by the Ordnance Survey of Ireland in his late teens in 1839, and moved to work for the Ordnance Survey for Great Britain in 1842. In the decade of the 1840s, a railroad-building boom was in progress, and Tyndall's land surveying experience was valuable and in demand by the railway companies. Between 1844 and 1847, he was lucratively employed in railway construction planning.

In his lectures at the Royal Institution Tyndall put a great value on, and was talented at producing, lively, visible demonstrations of physics concepts. In one lecture, Tyndall demonstrated the propagation of light down through a stream of falling water via total internal reflection of the light. It was referred to as the "light fountain". It is historically significant today because it demonstrates the scientific foundation for modern fibre optic Technology. During second half of the 20th century Tyndall was usually credited with being the first to make this demonstration. However, Jean-Daniel Colladon published a report of it in Comptes Rendus in 1842, and there's some suggestive evidence that Tyndall's knowledge of it came ultimately from Colladon and no evidence that Tyndall claimed to have originated it himself.

In 1847 Tyndall opted to become a mathematics and surveying Teacher at (Queenwood College), a boarding school in Hampshire. Recalling this decision later, he wrote: "the Desire to grow intellectually did not forsake me; and, when railway work slackened, I accepted in 1847 a post as master in Queenwood College." Another recently arrived young Teacher at Queenwood was Edward Frankland, who had previously worked as a chemical laboratory assistant for the British Geological Survey. Frankland and Tyndall became good friends. On the strength of Frankland's prior knowledge, they decided to go to Germany to further their education in science. Among other things, Frankland knew that certain German universities were ahead of any in Britain in experimental chemistry and physics. (British universities were still focused on classics and mathematics and not laboratory science.) The pair moved to Germany in summer 1848 and enrolled at the University of Marburg, attracted by the reputation of Robert Bunsen as a Teacher. Tyndall studied under Bunsen for two years. Perhaps more influential for Tyndall at Marburg was Professor Hermann Knoblauch, with whom Tyndall maintained communications by letter for many years afterwards. Tyndall's Marburg dissertation was a mathematical analysis of screw surfaces in 1850 (under Friedrich Ludwig Stegmann). Tyndall stayed in Germany for a further year doing research on magnetism with Knoblauch, including some months' visit at the Berlin laboratory of Knoblauch's main Teacher, Heinrich Gustav Magnus. It is clear today that Bunsen and Magnus were among the very best experimental science instructors of the era. Thus, when Tyndall returned to live in England in summer 1851, he probably had as good an education in experimental science as anyone in England.

An index of 19th century scientific research journals has John Tyndall as the author of more than 147 papers in science research journals, with practically all of them dated between 1850 and 1884, which is an average of more than four papers a year over that 35-year period.

Tyndall visited the Alps mountains in 1856 for scientific reasons and ended up becoming a pioneering mountain climber. He visited the Alps almost every summer from 1856 onward, was a member of the very first mountain-climbing team to reach the top of the Weisshorn (1861), and lead of one of the early teams to reach the top of the Matterhorn (1868). He is one the names associated with the "Golden age of alpinism", i.e. the mid-Victorian years when the more difficult of the Alpine peaks were summited for the first time.

Tyndall's three longest tutorials, namely Heat (1863), Sound (1867), and Light (1873), represented state-of-the-art experimental physics at the time they were written. Much of their contents were recent major innovations in the understanding of their respective subjects, which Tyndall was the first Writer to present to a wider audience. One caveat is called for about the meaning of "state of the art". The books were devoted to laboratory science and they avoided mathematics. In particular, they contain absolutely no infinitesimal calculus. Mathematical modelling using infinitesimal calculus, especially differential equations, was a component of the state-of-the-art understanding of heat, light and sound at the time.

Many of his readers interpret Tyndall to be a confirmed agnostic, though he never explicitly declared himself to be so. The following statement from Tyndall is an Example of Tyndall's agnostic mindset, made in 1867, and reiterated in 1878: "The phenomena of matter and force come within our intellectual range... but behind, and above, and around us the real mystery of the universe lies unsolved, and, as far as we are concerned, is incapable of solution.... Let us lower our heads, and acknowledge our ignorance, priest and Philosopher, one and all."

Tyndall became financially well-off from sales of his popular books and fees from his lectures (but there is no evidence that he owned commercial patents). For many years he got non-trivial payments for being a part-time scientific advisor to a couple of quasi-governmental agencies and partly donated the payments to charity. His successful lecture tour of the United States in 1872 netted him a substantial amount of dollars, all of which he promptly donated to a trustee for fostering science in America. Late in life his money donations went most visibly to the Irish Unionist political cause. When he died, his wealth was £22,122. For comparison's sake, the income of a police constable in London was about £80 per year at the time.

Though not nearly so prominent as Huxley in controversy over philosophical problems, Tyndall played his part in communicating to the educated public what he thought were the virtues of having a clear separation between science (knowledge & rationality) and religion (faith & spirituality). As the elected President of the British Association for the Advancement of Science in 1874, he gave a long keynote speech at the Association's annual meeting held that year in Belfast. The speech gave a favourable account of the history of evolutionary theories, mentioning Darwin's name favourably more than 20 times, and concluded by asserting that religious sentiment should not be permitted to "intrude on the region of knowledge, over which it holds no command". This was a hot topic. The newspapers carried the report of it on their front pages – in the British Isles, North America, even the European Continent – and many critiques of it appeared soon after. The attention and scrutiny increased the friends of the evolutionists' philosophical position, and brought it closer to mainstream ascendancy.

Tyndall did not marry until age 55. His bride, Louisa Hamilton, was the 30-year-old daughter of a member of parliament (Lord Claud Hamilton, M.P.). The following year, 1877, they built a summer chalet in the Swiss Alps. Before getting married Tyndall had been living for many years in an upstairs apartment at the Royal Institution and continued living there after marriage until 1885 when a move was made to a house near Haslemere 45 miles southwest of London. The marriage was a happy one and without children. He retired from the Royal Institution at age 66 having complaints of ill health.

Tyndall was an experimenter and laboratory apparatus builder, not an abstract model builder. But in his experiments on radiation and the heat-absorptive power of gases, he had an underlying agenda to understand the physics of molecules. Tyndall said in 1879: "During nine years of labour on the subject of radiation [in the 1860s], heat and light were handled throughout by me, not as ends, but as instruments by the aid of which the mind might perchance lay hold upon the ultimate particles of matter." This agenda is explicit in the title he picked for his 1872 book Contributions to Molecular Physics in the Domain of Radiant Heat. It is present less explicitly in the spirit of his widely read 1863 book Heat Considered as a Mode of Motion. Besides heat he also saw magnetism and sound propagation as reducible to molecular behaviours. Invisible molecular behaviours were the ultimate basis of all physical activity. With this mindset, and his experiments, he outlined an account whereby differing types of molecules have differing absorptions of infrared radiation because their molecular structures give them differing oscillating resonances. He'd gotten into the oscillating resonances idea because he'd seen that any one type of molecule has differing absorptions at differing radiant frequencies, and he was entirely persuaded that the only difference between one frequency and another is the frequency. He'd also seen that the absorption behaviour of molecules is quite different from that of the atoms composing the molecules. For Example, the gas nitric oxide (NO) absorbed more than a thousand times more infrared radiation than either nitrogen (N₂) or oxygen (O₂). He'd also seen in several kinds of experiments that – no matter whether a gas is a weak absorber of broad-spectrum radiant heat – any gas will strongly absorb the radiant heat coming from a separate body of the same type of gas. That demonstrated a kinship between the molecular mechanisms of absorption and emission. Such a kinship was also in evidence in experiments by Balfour Stewart and others, cited and extended by Tyndall, that showed with respect to broad-spectrum radiant heat that molecules that are weak absorbers are weak emitters and strong absorbers are strong emitters. (For Example, rock-salt is an exceptionally poor absorber of heat via radiation, and a good absorber of heat via conduction. When a plate of rock-salt is heated via conduction and let stand on an insulator, it takes an exceptionally long time to cool down; i.e., it's a poor emitter of infrared.) The kinship between absorption and emission was also consistent with some generic or abstract features of resonators. The chemical decomposition of molecules by lightwaves (photochemical effect) convinced Tyndall that the resonator could not be the molecule as a whole unit; it had to be some substructure, because otherwise the photochemical effect would be impossible. But he was without testable ideas as to the form of this substructure, and did not partake in speculation in print. His promotion of the molecular mindset, and his efforts to experimentally expose what molecules are, has been discussed by one Historian under the title "John Tyndall, The Rhetorician of Molecularity".

In Rome the Pope in 1864 decreed that it was an error that "reason is the ultimate standard by which man can and ought to arrive at knowledge" and an error that "divine revelation is imperfect" in the Bible – and anyone maintaining those errors was to be "anathematized" – and in 1888 decreed as follows: "The fundamental doctrine of rationalism is the supremacy of the human reason, which, refusing due submission to the Divine and eternal reason, proclaims its own independence.... A doctrine of such character is most hurtful both to individuals and to the State.... It follows that it is quite unlawful to demand, to defend, or to grant, unconditional [or promiscuous] freedom of thought, speech, writing, or religion." Those principles and Tyndall's principles were profound enemies. Luckily for Tyndall he didn't need to get into a contest with them in Britain, nor in most other parts of the world. Even in Italy, Huxley and Darwin were awarded honorary medals and most of the Italian governing class was hostile to the papacy. But in Ireland during Tyndall's lifetime the majority of the population grew increasingly doctrinaire and vigorous in its Roman Catholicism and also grew stronger politically. It would have been a waste of time for Tyndall to debate the Irish Catholics, but between 1886 and 1893 Tyndall was active in the debate in England about whether to give the Catholics of Ireland more freedom to go their own way. Like the great majority of Irish-born Scientists of the 19th century he opposed the Irish Home Rule Movement. He had ardent views about it, which were published in newspapers and pamphlets. For Example, in an opinion piece in The Times on 27 December 1890 he saw Priests and Catholicism as "the heart and soul of this movement" and wrote that placing the non-Catholic minority under the dominion of "the priestly horde" would be "an unspeakable crime". He tried unsuccessfully to get the UK's premier scientific society to denounce the Irish Home Rule proposal as contrary to the interests of science.

In his last years Tyndall often took chloral hydrate to treat his insomnia. When bedridden and ailing, he died from an accidental overdose of this drug in 1893 at the age of 73, and was buried at Haslemere. Afterwards, Tyndall's wife took possession of his papers and assigned herself supervisor of an official biography of him. She dragged her feet on the project, however, and it was still unfinished when she died in 1940 aged 95. The book eventually appeared in 1945, written by A. S. Eve and C. H. Creasey, whom Louisa Tyndall had authorised shortly before her death.

The majority of the progressive and innovative British physicists of Tyndall's generation were conservative and orthodox on matters of religion. That includes for Example James Joule, Balfour Stewart, James Clerk Maxwell, George Gabriel Stokes and william Thomson – all names investigating heat or light contemporaneously with Tyndall. These conservatives believed, and sought to strengthen the basis for believing, that religion and science were consistent and harmonious with each other. Tyndall, however, was a member of a club that vocally supported Darwin's theory of evolution and sought to strengthen the barrier, or separation, between religion and science. The most prominent member of this club was the Anatomist Thomas Henry Huxley. Tyndall first met Huxley in 1851 and the two had a lifelong friendship. Chemist Edward Frankland and Mathematician Thomas Archer Hirst, both of whom Tyndall had known since before going to university in Germany, were members too. Others included the social Philosopher Herbert Spencer.