Born in Potsdam, Germany, Helmholtz studied medicine at the Friedrich Wilhelm Institute in Berlin and obtained his MD in 1842. He returned to Potsdam to become an army surgeon, but returned to civilian life in 1848 and was appointed assistant at the Anatomical Museum in Berlin. He then held a succession of chairs at Königsberg (1849–55), Bonn (1855–58), Heidelberg (1858–71), and Berlin (1871–77) and later became director of the Physico-Technical Institute at Berlin Charlottenburg.

Helmholtz made major contributions to two areas of science: physiology and physics. In physiology he invented (1851) the ophthalmoscope for inspecting the interior of the eye and the ophthalmometer for measuring the eye's curvature. He investigated accommodation, color vision, and color blindness. His book Handbuch der physiologische Optik (Handbook of Physiological Optics) was published in 1867. Helmholtz also worked on hearing, showing how the cochlea in the inner ear resonates for different frequencies and analyzes complex sounds into harmonic components. In 1863 he published Die Lehre von den Tönemfindungen als physiologische Grundlage für die Theorie der Musik (The Sensation of Tone as a Physiological Basis for the Theory of Music). Another achievement was his measurement of the speed of nerve impulses (1850).

One of Helmholtz's interests had been muscle action and animal heat and this, inspired by his distaste for vitalism, led him to his best-known discovery – the law of conservation of energy. This was developed independently of the work of James Joule and Julius von Mayer and published as Über die Erhaltung der Kraft (1847; On the Conservation of Force). He showed that the total energy of a collection of interacting particles is constant, and later applied this idea to other systems.

Helmholtz also worked in thermodynamics, where he introduced the concept of free energy (energy available to perform work). In electrodynamics he attempted to produce a general unified theory. Heinrich Hertz, who discovered radio waves in 1888, was Helmholtz's pupil.