Abstract: tert-Butanol，as a novel reaction medium，was adopted for lipase-catalyzed trans-esterification of rapeseed oil for bio-diesel production，and the negative effects caused by excessive methanol and by-product glycerol could be eliminated.When the water content reached 2% or phospholipids content was more than 0.1% based on the oil mass，there would be obvious negative effects on lipase LipozymeIM TL and Novozym 435.Combined use of LipozymeIM TL and Novozym 435 was proposed further to catalyze the methanolysis，and the water-resistant and phospholipids-resistant abilities of the lipases could be improved.