Moseley came from an academic family in Weymouth. He graduated in natural sciences from Oxford University in 1910 and then joined Ernest Rutherford at Manchester University to work on radioactivity, although he soon turned his attention to x-ray spectroscopy. He returned to Oxford in 1913 to continue his work under J. S. E. Townsend.

When x-rays are produced by an element a continuous spectrum is emitted together with a more powerful radiation of a few specific wavelengths characteristic of the element. To investigate the positive charge on atomic nuclei Moseley examined these characteristic spectral lines using crystal diffraction. For a number of elements, he discovered a regular shift in the lines with increasing atomic weight. From this he determined for each element an integer approximately proportional to the square root of the frequency of one of its spectral lines. This integer, now called the atomic number (or proton number), equaled the positive charge on the atomic nuclei. Moseley's work led to major improvements in Dmitri Mendeleev's periodic table and enabled elements to be classified in a new and more satisfactory manner.

At the outbreak of World War I Moseley enlisted in the army and was commissioned in the Royal Engineers. His death, from a sniper's bullet at Gallipoli, cut short what promised to be a most brilliant career in science.