Abstract In addition to being an essential natural amino acid, l-histidine is biologically important in the dismutation of superoxide radical (O 2 − ) by superoxide dismutase (SOD). In this work, fluorescence and absorptiometric techniques were used to characterize the photo-phenomenon and optical properties of this compound in a simulated body fluid (SBF). l-Histidine fluoresces at 360 nm when excited at 220 nm. Its molar absorptivity, ɛ, is 4.8 × 10 3 M −1 cm −1. The observed bimolecular quenching rate constant, k q, of 7.5 × 10 8 M −1 s −1, by hydrogen peroxide, suggests a non-diffusional activation-controlled mechanism with a rate constant, k a, of 8.55 × 10 8 M −1 s −1 and an electron transfer rate constant, k ET of 6.06 × 10 8 s −1. The determined radiative and non-radiative rate constants, 4.73 × 10 7 and 2.9 × 10 8 s −1, respectively, suggests that the deactivation of the thermally excited l-histidine is by non-radiative route rather than by normal fluorescence, which accounts for the low quenching constant, K SV, of 2.22 M −1 that was obtained. The solvent reorganization energy, λ s, and the reaction free energy change, Δ G, of 1.48 and −5.62 eV, respectively, suggest that the electron transfer reaction in the l-histidine–H 2O 2 reaction is through a solvent separated mechanism.