Abstract: An orthogonal experimental design was used to optimize the protein extraction technology for Grifola frondosa. The results showed that the influence of extraction factors on the index was decreased in order C > D > B > A. The optimal extraction technology included, the ratio of material to liquid was 1∶50, the extracting temperature was 45℃, the pH value was 10, and 5 times of the cells ultrasonic crush, and under these conditions, the extraction rate for protein of Grifola frondosa reached 0.95%.