Barkla was born in Widnes in the northwest of England. After taking his master's degree in 1899 at Liverpool, Barkla went to Trinity College, Cambridge but, because of his passion for singing, he transferred to King's College to sing in the choir. At King's College he started his important research on x-rays. In 1902 he returned to Liverpool as Oliver Lodge Fellow and in 1909 became Wheatstone Professor at King's College, London. From 1913 onward he was professor of natural philosophy at Edinburgh University.

His scientific work, for which he received the 1917 Nobel Prize for physics, concerned the properties of x-rays – in particular, the way in which they are scattered by various materials. He showed in 1903 that the scattering of x-rays by gases depends on the molecular weight of the gas. In 1904 he observed the polarization of x-rays – a result that indicated that x-rays are a form of electromagnetic radiation like light. Further confirmation of this was obtained in 1907 when he performed certain experiments on the direction of scattering of a beam of x-rays as evidence to resolve a controversy with William Henry Bragg who argued, at the time, that x-rays were particles.

Barkla also demonstrated x-ray fluorescence, in which primary x-rays are absorbed and the excited atoms then emit characteristic secondary x-rays. The frequencies of the characteristic x-rays depend on the atomic number of the element, as shown by Henry Moseley, who could well have shared Barkla's Nobel Prize but for his untimely death.

From about 1916, Barkla became isolated from modern physics with an increasingly dogmatic attitude, a tendency to cite only his own papers, and a concentration on untenable theories.