Abstract: Tert-amyl alcohol was used as a reaction medium for Novozym 435 lipase-catalyzed methanolysis of soybean oil for biodiesel production. With tert-amyl alcohol as reaction medium，both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. The highest biodiesel yield of 97% was obtained under the optimum conditions (tert-amyl alcohol/oil volume ratio 1，methanol/oil molar ratio 3，2% Novozym 435 based on the soybean oil weight，temperature 40 ℃，2% water content based on soybean oil，180 r/min and 15 h). Moreover，there was no evident loss in lipase activity and stability even after being repeatedly used for 160 runs and the yield of biodiesel production was more than 95%.