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Kirchhoff studied in his native city of Königsberg (now Kaliningrad in Russia), graduating in 1847. Three years later he was appointed a professor at Breslau. He moved to Heidelberg, where Robert Bunsen was professor of chemistry, in 1854.

Kirchhoff was one of the foremost physicists of the 19th century and is remembered as one of the founders of the science of spectroscopy. He is also known for Kirchhoff's laws, formulated in 1845 while he was still a student, which refer to the currents and electromotive forces in electrical networks.

In 1859 he published an explanation of the dark lines in the solar spectrum discovered by Josef von Fraunhofer, in which he suggested that they are due to absorption of certain wavelengths by substances in the Sun's atmosphere. He later formulatedKirchhoff's law of radiation, which concerns the emission and absorption of radiation by a hot body. It states that the rate of emission of energy by a body is equal to the rate at which the body absorbs energy (both emission and absorption being in a given direction at a given wavelength). Kirchhoff gave a final proof of this in 1861.

In about 1860 Bunsen was analyzing the colors given off by heating chemicals to incandescence, using colored glass to distinguish between similar shades. Kirchhoff joined this research when he suggested that the observation of spectral lines, by dispersing the light with a prism, would be a more precise way of testing the color of the light. Kirchhoff and Bunsen found that each substance emitted light that had its own unique pattern of spectral lines – a discovery that began the spectroscopic method of chemical analysis. In 1860, a few months after publishing these results, they discovered a new metal, which they called cesium, and the next year found rubidium. Kirchhoff and Bunsen also constructed improved forms of the spectroscope for such work and Kirchhoff showed that, if a gas emitted certain wavelengths of light then it would absorb those wavelengths from light passing through it.

Kirchhoff was crippled by an accident in mid-life but remained in good spirits and, when his health forced him to stop experimental work in 1875, he was offered the chair of theoretical physics in Berlin. He remained there until his death 12 years later.