The aim of this paper is to show the chemical and physical basis of bioluminescence. Bioluminescence (BL) is a phenomenon of light emission of living organisms, such as fireflies, different sea creatures, bioluminescent bacterias and fungi. BL is a chemical process in nature caused by the interaction of a protein, luciferase, and its substrate luciferin. The emitted light has different maximum wavelengths and therefore different colors of emitted light can be noticed. Bioluminescence contains, in essence, the same principles as chemiluminescence. As with any chemical reaction, it is desirable to know the kinetics of the BL reaction, which agrees with the kinetics of biochemical reactions, ie follows the Michaelis-Menten kinetics. Knowledge of quantum yields (quantum efficiency) is of great importance in the application of BL because with increasing quantum yield the limit of detection decreases. The structures of 9 luciferins are known, and five BL systems, along D-luciferin metabolism have been described. The main bioluminescent component does not have to be a luciferase-luciferin pair but also photoproteins, of which obelin and aequorin are the most important. Luciferases are of great importance today for application in medicine, so it is desirable to know their structure, advantages and disadvantages, and if necessary, modify their luciferins to be as effective as possible. Bioluminescence finds its greatest application in bioluminescent imaging, which facilitates tumor detection, observation of disease progression, and drug efficacy.