Besides high cost, the most important reasons that immobilized lipases are limited in industrialization of biodiesel production are the toxicity of methanol and the adsorption of glycerol onto the surface of immobilized vector. Solvent engineering method was employed to the reaction where compound-lipase with synergistic effect. Novozym 435 and Lipozyme TL IM, catalyzed preparation of biodiesel from stillingia oil with methanol. The treatment accelerated the solubility of methanol in oil and dissolved glycerol, which helped maintain lipase activity. It is found that the yields of biodiesel in co-solvent exceeded those in the pure organic solvents. The mixture system of co-solvent with 60% acetonitrile and 40% t-butanol (v/v) was proved to be an optimal one, and RSM was used to optimize the reaction factors and the optimal conditions: methanol/oil molar ratio 6.4:1, compound-lipase 4.32% (wt/wt) and molecular sieve 5.5% (wt/wt). R-2-=98.86% showed good coincidence between predicted and experimental values. There was nearly no loss inactivity of compound-lipase after being recycled for 30 times. Other oils were also investigated in the mixture system, and we got the same results, which indicated that the mixture system could be an ideal prospective medium applied to biodiesel production. (C) 2010 Elsevier B.V. All rights reserved.