In order to seek the optimal condition for preparing biodiesel from waste oil by biological enzyme, the condition of transesterification was optimized by response surface methodology. Various molar ratio of methanol to oil, enzyme amount, reaction temperature and reaction time selected as the independent variable, and the yield of biodiesel as response value, the experiment with 4 factors and 3 levels was designed by Box-Benhken central composite, and after data preprocessing a regression model was founded by response surface analysis. Analysis showed that the result of the regression equation obtains good fitting, the optimal condition of the transesterification were: molar ratio of methanol to oil 4∶1, enzyme amount 10%, reaction temperature 35 ℃ and reaction time 15 h. Under these conditions, the yield of biodiesel was 95.9%.