Abstract: Biodiesel production from renewable biological resources won wide attention of the world. The immobilized enzyme Novo435 was used as catalyst to catalyze the transesterification reaction of rapeseed oil with methanol in non-solvent system. The effects of variable in the reaction such as the molar ratio of methanol to oil, the amount of catalyst, the reaction time, the reaction temperature, the rate of rotation and the water content were investigated respectively, The optimum conditions of the transesterification reaction are the rotation rate 200 r/min, the molar ratio of methanol to oil 1.5∶1, the reaction temperature 50℃ and lipase amount 10%(by the weight of the oil). Under the conditions, 47% of the rapeseed oil was converted to its corresponding methyl esters. Water is an unfavorable factor in the transesterification reaction. When the water exists in the reaction system, the conversion of the oil decreased to 30%. The methanol can be added by two steps, 80% of the ester conversion was achieved after reacting for 26 h.