Horseradish Peroxidase(HRP) is a widely used biocatalyst. However, activity change mechanism of HRP after irradiation has not been reported. In this research, the effects of irradiation on the catalytic activity of HRP and the mechanism of enzyme activity were studied by UV-Vis absorption、synchronous fluorescence and circular dichroism(CD) spectroscopy. The experimental results showed that irradiation could promote HRP reduction, while its catalytic activity would change. The UV-Vis absorption spectra date showed that the maximum absorption peak of the Soret band of HRP was red shift after irradiation and the oxidation peak intensity at 498 nm was decreased, and consequently HRPFe(Ⅲ) was reduced to HRPFe(Ⅱ). The effect sequence of wavelength on the photoreduction was 280 nm> 254 nm> 498 nm> 403 nm; the activity of the enzyme was 403 nm> 498 nm> 254 nm> 280 nm; while irradiated by 280 nm light , Phe, Trp, Tyr, Cys free amino acids and glutathioneall could promote the photoreduction process. Enzyme activity was closely related to the degree of photoreduction, and Fe(Ⅱ) could not provide empty orbit coordination with H2O2, so light-induced reduction of Fe(Ⅲ) led to decreased activity. Synchrotron fluorescence and circular dichroism spectroscopy revealed that the conformation of HRP did changed after photoreduction, so the conformational change was the reason for the change of enzyme activity. The factors that affect the decrease of enzyme activity by ultraviolet light are the contribution of light-induced active center Fe(Ⅲ) reduction and protein conformational changes. The results of this study have important theoretical and practical significance for a further understanding of the effects of light on hemoglobin(enzyme) structures and functions.