Abstract: Objective: To enhance the liquid-submerged fermentation biosynthesis of ergothioneine (EGT) in Pleurotus ostreatus. Methods: The influence of different addition methods of corn steep liquor hydrolysate (CSLH) combined with amino acids on the EGT content in liquid-submerged fermentation biosynthesis was investigated through single-factor experiments. Using EGT content as the evaluation index, the response surface Box-Behnken experimental design was employed to optimize the optimal addition amount of combined amino acids. Results: The EGT content in the fermented product increased by 26.26% when 1.5% CSLH was added. CSLH had a synergistic effect on the synthesis of EGT with histidine (His), cysteine (Cys) and methionine (Met). The EGT content in the fermentation reached 87.52 mg/L when 1.5% CSLH was used in combination with 7.7 mmol/L His, 10.0 mmol/L Cys, and 9.5 mmol/L Met, which was 2.53 times the content with only 1.5% CSLH added, and 5.13 times the content without CSLH and amino acids added. Conclusion: CSLH, as a growth promoter, in synergy with His, Cys, and Met, can effectively enhance the biosynthesis of EGT, providing a new regulatory strategy for industrial fermentation production of EGT.