Crookes studied at the Royal College of Chemistry in his native city of London, under August von Hofmann (1848). After working at the Radcliffe Observatory, Oxford, and the Chester College of Science, he returned to London in 1856, where, having inherited a large fortune, he edited Chemical News and spent his time on research.

Following the invention of the spectroscope by Robert Bunsen and Gustav Kirchhoff, Crookes discovered the element thallium (1861) by means of its spectrum. In investigating the properties and molecular weight of thallium, he noticed unusual effects in the vacuum balance that he was using. This led him to investigate effects at low pressure and eventually to invent the instrument known as the Crookes radiometer (1875). This device is a small evacuated glass bulb containing an arrangement of four light metal vanes. Alternate sides of the vanes are polished and blackened. When radiant heat falls on the instrument, the vanes rotate. The effect depends on the low pressure of gas in the bulb; molecules leaving the dark (hotter) surfaces have greater momentum than those leaving the bright (cooler) surfaces. Although the instrument had little practical use, it was important evidence for the kinetic theory of gases.

Crookes went on to investigate electrical discharges in gases at low pressure, producing an improved vacuum tube (the Crookes tube). He also investigated cathode rays and radioactivity. Crookes glass is a type of glass invented to protect the eyes of industrial workers from intense radiation.

From about 1870, Crookes became interested in spiritualism and became one of the leading investigators of psychic phenomena.