Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores).

The most commonly observed autofluorescencing molecules are NADPH and flavins; the extracellular matrix can also contribute to autofluorescence because of the intrinsic properties of collagen and elastin.

Generally, proteins containing an increased amount of the amino acids tryptophan, tyrosine, and phenylalanine show some degree of autofluorescence.

Autofluorescence also occurs in non-biological materials found in many papers and textiles. Autofluorescence from U.S. paper money has been demonstrated as a means for discerning counterfeit currency from authentic currency.