Photoinduced formation of protein-flavin adducts is crucial in photoresponsive proteins containing light-oxygen-voltage (LOV) domains. LOV proteins typically share an N-terminal sensor domain with FMN and a C-terminal effector domain such as a kinase, a phosphodiesterase, or a DNA-binding protein. Light absorption by FMN results in a covalent flavin-cysteine adduct, which allosterically translates to the linked effector domain. Photoinduced protein-flavin adducts have not been reported in enzymes not involved in light-dependent processes. Here, we have used fluorescence, pH effects, and mutagenesis to follow up a serendipitous observation of an unusual fluorescence excitation spectrum in choline oxidase at alkaline pH (M. Ghanem and G. Gadda, unpublished observations). Physiologically, choline oxidase oxidizes choline to betaine through two FAD-associated reactions and is not a photoenzyme. The enzyme-bound flavin showed a progressive shift of the fluorescence excitation maximum (λex) from 468 to 399 nm with increasing pH values between pH 6.0 and 10.0, consistent with a metastable photoinduced protein-flavin adduct. In contrast, the maximal λem was independent of pH, with values of ∼526 nm. For comparison, fluorescence spectra of FAD in bulk solution had maximal values differing by ≤2 nm at different pH values, with λex at 453 nm and λem at 527 nm. The unusual behavior of the enzyme persisted in the mutated S101A enzyme variant but was eliminated in the H466Q variant, suggesting that the photoinduced species is likely a C4a-N-histidyl-FAD. The results provide evidence that metastable photoinduced formation of a flavin-protein adduct can occur in an enzyme that is not a photoreceptor or a photoenzyme.