Abstract: Glycerophosphorylcholine was prepared by hydrolysis of soybean concentrated phospholipids using complex enzyme as catalyst in the present study.The HPLC standard curve of glycerophosphorylcholine(GPC) used for the qualitative and quantitative analysis was established,and the equation was y=8 179.1x-3 492.1(R²=0.998 3).The contents of phospholipids and phosphatidylcholine in the raw material were determined,which were 42%and 10.4%,respectively.The effect of reaction time,reaction temperature and enzyme loading ratio on the preparation of GPC by complex enzymatic method were investigated by single factor experiments.Response Surface Methodology(RSM) adopting Box-Benhnken Design(BBD) principle was used to further optimize the preparation processing conditions of GPC on the basis of single factor experiment,with the conversion rate of GPC as evaluation indicator.The optimal conditions for GPC preparation were as follows:reaction time 95 min,reaction temperature 41℃and enzyme loading ratio 1.1%.Under these conditions,the conversion rate of GPC reached to 70.65%,which was nearly consistent with the predicted value.