OBJECTIVE： To optimize the ultrasonic extraction technology of polyphenol from the stem of Trapa natans and conduct in vitro antitumor activity test. METHODS： Using the yield of polyphenol as dependent variable， ethanol concentration， extraction time and liquid-material ratio as independent variables， the ultrasonic extraction technology of polyphenol from the stem of T. natans was optimized by response surface methodology designed by Box-Behnken design. The effects of different concentrations of polyphenol from the stem of T. natans on the proliferation of HeLa cells， U251 cells and HepG2 cells were detected by MTT assay， IC50 were calculated，and morphologic changes of them were observed. RESULTS： The optimal condition as follows as the liquid-material ratio  was 34，45％ ethanol， ultrasonic extracting for 24 min at 50 ℃. Under this condition， the yield of polyphenol was 29.36 mg/g （RSD＝0.72％，n＝3）， the relative error of which from predicted value 29.89 mg/g was 1.77％. The proliferation of HeLa cells， U251 cells and HepG2 tumor cells were all significantly inhibited by polyphenol with IC50 of 166.2， 141.7， 126.6    μg/ml， showing good dose-effect relationship. The apoptosis of those cells was presented to certain extent. CONCLUSIONS： Optimized ultrasonic extraction technology of polyphenol from the stem of T. natans by response surface methodology is simple and predictable. The polyphenol from the stem of T. natans has significant anti-tumor effect in vitro.