[Objective] To optimize the process for transesterification reaction in biodiesel production by the synergistic biocatalyst in high water content system. [Methods] The synergistic biocatalyst of Candida antarctica lipase B (CALB) and Rhizopus oryzae lipase (ROL) were expressed and secreted in Pichia pastoris using genetic engineering techniques. The single factor experiment was used to optimize the process conditions, and the methyl ester (ME) yield was used to evaluate the transesterification ability of synergistical biocatalyst. [Results] The maximum ME yield of 93% could be achieved using 16 U/g oil combined with mixed lipase (CALB:ROL=7:3) for the methanolysis. The molar ratio of methanol to oil is 4:1 (initial methanol/oil molar ratio 2:1 and 1:1 at 12 h and 24 h, respectively), water content 30%?60% (W/W) (based on oil weight), temperature 40 °C for 29?34 h. [Conclusion] This synergistic catalytic system is environmental friendly. Furthermore, both the consumption of lipase and reaction time were decreased more than 50% compared with the conventional enzymatic preparation of biodiesel. Therefore, this study provides an effective way to decrease the cost of biodiesel preparation and also has great potential in biodiesel industry.