The present invention belongs to the technical field of genetic engineering and fermentation engineering, and in particular relates to a method for preparing S-lactoylglutathione, wherein glutamate, glycine, cysteine and methylglyoxal are used as raw materials, and are converted into S-lactoylglutathione under the catalysis of glutathione synthetase and glyoxalase. According to the present invention, the raw materials with relatively low cost are used for fermentation, the operation is simple, the conversion rate is high, the yield of the prepared S-lactoylglutathione is high, and the method is suitable for batch industrial production.