A new technique of biodiesel production from Sapium sebiferum oil catalyzed by immobilized lipase from Pseudomonas cepacia G63 prepared in our laboratory was investigated in this study. The independent factors were studied and the significant factors to the yield of biodeisel were confirmed, and the Box-Behnken design was employed to evaluate the effects of those significant factors on yield in preparation of biodiesel. Results indicated the optimal condition for biodiesel preparation were: 4:1 methano/oil molar ratio, 2.7% (w/w) lipase, temperature 41 degrees C, and the subsequent verification experiment got a result of (96.22%) kept coincided with the predicted biodiesel yield (97.07%) under the optimal conditions, and R(2) = 98.19% shown the model was considered to be accurate and available for predicting the yield of biodiesel. There was no loss nearly in the immobilized lipase activity after being repeatedly used for 20 cycles at the optimal reaction condition. (C) 2010 Elsevier Ltd. All rights reserved.