Candida rugosa lipase and Ryzopus oryzae lipase were simultaneously immobilized on silica gel following enzyme pretreatment. The factors affecting the co-immobilization process, such as reaction time and enzyme ratio, were investigated. Biodiesel was then produced by using the co-immobilized enzyme matrix. A batch system was employed with stepwise methanol feeding, and the continuous process involved a packed-bed reactor. Under optimal immobilization conditions, the activity was approximately 16,000 U/g·matrix. When co-immobilized enzyme was used with optimized stepwise methanol feeding, conversion of biodiesel reached about 99% at 3 h and was maintained at a level of over 90% for about 30 reuses.