Ultrasonic-assisted extraction was applied to extract oxyresveratrol from cultured black mulberry bark (OCBMB). The process was optimized by response surface methodology. According to the results of the signal factor test, the Box-Behnken design was employed to investigate the effects of four chosen independent variables (ultrasonic power, extraction time, ethanol concentration, and acetic acid concentration) on the oxyresveratrol extraction, and the response surface plots showed that the parameters exhibited interactive effects. The optimal conditions were as follows: extraction temperature, 50 degrees C; ratio of solution to solid (v/m), 20:1; ultrasonic power, 496.23W; extraction time, 113min; ethanol concentration, 70.98%; acetic acid concentration, 0.77%. Under these optimal conditions, experimental values well matched with the predicted values, which indicated the model used had extremely good fitting degree and the response surface methodology for optimizing Ultrasonic-assisted extraction of OCBMB was successful.