Abstract: Immobilized lipase from Candida sp.99-125 was used for the synthesis of biodiesel from waste oil in organic solvent system. The effect of various factors on the reaction was studied, including the inhibition of short chain alcohols on enzymatic reaction, the nature of solvents, substrate concentration, water content, mode of adding methanol and so on. As can be seen from the results, the conversion rate reached 92% under the optimum conditions, and the life span of the immobilized lipase reached more than 7 batches.