Abstract: This work reported a novel technology on bioconversion of phosphatidylcholine (PC) into phosphatidylserine (PS) by phospholipase D (PLD) in an aqueous phase catalytic system through immobilization of substrate phosphatidylcholine (PC). The major factors affecting PC adsorption efficiency were investigated to obtain the optimal parameters, such as the kinds of precipitant reagents, carrier types, and substrate loading concentration. Furthermore, the time kinetics of PS catalytic synthesis by PLD through immobilization of substrate PC and the recycling utilization of enzyme PLD were also examined. Results showed that the yield of PS achieved 98.74% after 24 h reaction, and PLD had an excellent recycling utilization. When it was reused for 10 batches, PLD could still yield more than 60% of PS. In the end, the efficiency of PS catalytic synthesis by PLD was also compared between through immobilization of substrate PC and in two-liquid phase system. It was found that higher yield of PS and more excellent stability of PLD were achieved through immobilization of substrate PC than in two-liquid phase system. These findings would provide a novel catalytic system for PC bioconversion into PS in the future.