Weber was the eldest of three brothers who all made important contributions to science. He was born at Wittenberg in Germany and became a professor at the University of Leipzig in 1818, a position he held until his death.

Weber is best known for his work on sensory response to weight, temperature, and pressure. In 1834 he conducted research on the lifting of weights. From his researches he discovered that the experience of differences in the intensity of sensations depends on percentage differences in the stimuli rather than absolute differences. This is known as the just-noticeable difference (j.n.d.), difference threshold, or limen. The work was published in Der Tastsinn und das Gemeingefühl (1851; The Sense of Touch and the Common Sensibility) and was given mathematical expression by Weber's student Gustav Theodor Fechner as the Weber–Fechner law.

Weber is regarded as a founder of experimental psychology and psychophysics. He also conducted important anatomical work.