The alterations of papain activity and conformation were studied in condition of pretreatment with ethanol solutions at different concentrations, different pretreatment times, and in condition of various reaction concentrations. The result showed that the papain activity was obviously increased and the Km value was declined when treated with ethanol. Ultraviolet difference spectra showed that the secondary structure ofpapain altered in ethanol solutions. Fluorescence emission spectra showed that the peak of fluorescence emission was not shifted but the fluorescence intensity in papain increased in ethanol solutions.