A self-made lipase preparation from Candida sp. 99-125 was used for the production of biodiesel through enzymatic esterification of fatty acids. The crude lipase powder and fermentation broth were immobilized on a cheap fiber cloth carrier. The conditions of lipase-catalyzed esterification between long-chain fatty acids and methanol in a solvent system were investigated in detail, including the temperature, pH value, substrate concentration, solvent, absorbent agent, enzyme dosage and purity, immobilization method, the mode of addition of substrate. The results show that reaction temperature, pH of lipase micro-environment, substrate concentration, enzyme dosage and purity affect the esterification. strongly. Several new methods and enzymatic procedures for improving the enzymatic reaction involving the process cost are also discussed, such as fossil diesel fuel as reaction solvent, immobilization method, multi-step gradient addition of methanol. The esterification degree of 92.8% was obtained with oleic acid and methanol under the optimal reaction condition after 12.5 h reaction time. The half-life of the immobilized lipase preparation from crude free lipase powder for esterification was 15 days.