Abstract: In order to explore the optimal technological conditions for the production of glutathione (GSH) fermented by Saccharomyces cerevisiae, based on the single factor experiment, the liquid fermentation medium was optimized through Plackett-Burman design experiment combined with the response surface methodology. The results showed that the optimal culture medium for the production of GSH fermented by Saccharomyces cerevisiae was as follows: glucose of 71 g·L^-1, yeast extract paste of 4 g·L^-1, NH4Cl of 6 g·L^-1, KH₂PO₄ of 2.0 g·L^-1, MgSO₄ of 0.5 g·L^-1, pH of 6.0. After three batches of parallel experiments, the mean value for the actual yield of GSH was 80.5 mg·L^-1, which was similar to the predicted value and increased by about 19.2% higher than that before optimization (65.03 mg·L^-1).