Abstract: The interaction of puerarin and α-chymotrypsin was studied by fluorescence spectrometry,circular dichrosim spectrometry and measurement of enzyme activity.It was found that the mechanism of fluorescence quenching of α-chymotrypsin by puerarin was a static quenching of non-radiative energy transfer,and that the interaction between them was mainly related to the action of van der Waals forces or hydrogen bond.Distance between the binding position of puerarin in α-chymotrypsin and the tryptophan radical was calculated by the Frster non-radiative energy transfer theory and found to be 3.67 nm.Besides,change of secondary structure of α-chymotrypsin was induced by pueparin as shown by the result of circular dichroism spectroscopic study,and pueparin was capable to be used as an inhibitor for activity of α-chymotrypsin.