Abstract: Response surface methodology was used to optimize the ultrasonic-assisted extraction processing of protein from Agaricus bisporus. The preparation process of Agaricus bisporus protein was optimized according to Box-Benhnken central composition design principle on foundation of single factor experiment, the method of response surface analysis with 4 factors and 3 levels combined with Design-Expert 7. 0 data analysis software was adopted to study extraction temperature, liquid to solid ratio, ultrasonic power, ultrasonic time four factors and their interactions on extraction effect. According to the above results, the optimum preparation conditions were obtained as follows:ultrasonic power 130W, extraction temperature 43℃, ultrasonic time 40min, ratio of solid∶ liquid at 1∶ 5, the extraction of Agaricus bisporus protein reached 50. 39%.