Abstract: Podophyllotoxin (PPT) is a frequently used clinical natural drug. In this study, we aimed to optimize the extraction process of PPT from Diphylleia sinensis H. L. Li by response surface methodology (RSM). Based on single factor experiments, three important factors, that is, ethanol concentration, extraction temperature, and extraction time, were selected for further optimization with three-factor/three-level Box-Behnken design. Based on the extraction rate of PPT, the optimal conditions were:ethanol concentration of 48.18%, extraction temperature of 62.66℃, and extraction time of 16.15 h. Under these optimal conditions, PPT yield could reach 7.636%, with the confirmatory experiment reaching 7.54%. Thus, there was no significant difference compared with the theoretical value, verifying the optimized method with RSM in this study.