Born at Bekesbourne in Kent, Hales entered Cambridge University in 1696 to study theology. He was ordained in 1703 and appointed curate at Teddington, near London, in 1708 (or 1709). During his time at Cambridge, he studied science and was influenced by Isaac Newton's ideas, which still dominated scientific thought at the university and probably accounted for Hales's consistent use of the quantitative method in his biological researches.

Hales was elected a fellow of the Royal Society in 1718 but his first work, Vegetable Staticks, was not published until 1727. In this book, which included his most important observations in plant physiology, Hales demonstrated that plant leaves absorb air and that a portion of air is used in plant nutrition. In addition, he realized that light is necessary for growth and investigated growth rates by marking plants at regular intervals. He measured the rate of water loss (transpiration) in plants, finding that it occurred through the leaves and was responsible for an upward flow of sap in plants. From additional measurements of sap flow he concluded that there was no circular movement of sap in plants analogous to blood circulation in animals.

Hales also made important contributions to the understanding of blood circulation by measuring such properties as blood pressure, output per minute from the heart, rate of flow and resistance to flow in vessels. The results were published in Haemastaticks (1733; Blood Statics).

Other notable discoveries include the development of methods for collecting gases over water, distilling fresh water from sea water, and preserving foodstuffs with sulfur dioxide. He also invented a ventilator for introducing fresh air into prisons, ships, and granaries.