Boltzmann studied at the university in his native city of Vienna, where he received his doctorate in 1866. He held professorships in physics or mathematics at Graz (1869–73; 1876–79), Vienna (1873–76; 1894–1900; 1902–06), Munich (1889–93), and Leipzig (1900–02).

Boltzmann made important contributions to the kinetic theory of gases. He developed the law of equipartition of energy, which states that the total energy of an atom or molecule is, on average, equally distributed over the motions (degrees of freedom). He also produced an equation showing how the energy of a gas was distributed among the molecules (called the Maxwell–Boltzmann distribution).

Boltzmann also worked on thermodynamics, in which he developed the idea that heat, entropy, and other thermodynamic properties were the result of the behavior of large numbers of atoms, and could be treated by mechanics and statistics. In particular, Boltzmann showed that entropy – introduced by Rudolf Clausius – was a measure of the disorder of a system. Boltzmann's equation (1896) relates entropy (S) to probability (p): S = klog p + b. The constant k is known as Boltzmann's constant and has the value 1.380 54 × 10^-23 joule per kelvin. The equation is engraved on his gravestone.

Boltzmann's work in this field was heavily criticized by opponents of atomism, particularly Wilhelm Ostwald. It did however lead to the science of statistical mechanics developed later by Josiah Willard Gibbs and others. Boltzmann also worked on electromagnetism. He is further noted for a theoretical derivation of the law of radiation discovered by Josef Stefan.

Toward the end of his life he suffered from illness and depression, and committed suicide in 1906.