Abstract: To reduce the content of free gossypol in cottonseed meal and improve the quality of cottonseed protein, this study used a two-stage fermentation method to treat cottonseed meal. Enterococcus faecalis (E) was first added, and then Aspergillus oryzae (M1) was added for staged fermentation. The fermentation conditions of mixed were optimized by single factor and orthogonal experiments. The results showed that when the inoculation amount of Enterococcus faecium was 1.5%, the fermentation temperature was 37 ℃, the fermentation time was 24 h, the inoculation amount of Aspergillus oryzae was 1.5%, the fermentation temperature was 30 ℃, the fermentation time 96 h, and the initial fermentation moisture of cottonseed meal was 50%, the crude protein content of cottonseed meal increased from 39.6% to 49.64%, the acid-soluble protein content increased from 4.79% to 36.58%, and the free gossypol content decreased from 1370 mg/kg to 45.32 mg/kg, and the detoxification rate reached 96.7%. The results would provide a scientific basis for the high-value utilization of cottonseed meal.