The process of browning seriously influence the cider quality, but the glutathione producing by yeast can inhibit browning in the production and storage of cider. The glutathione (GSH) producing strain Saccharomyces cerevisiae Y18 was chosen as the starting strain, and after ultraviolet mutagenesis and diethyl sulfate mutagenesis, the strain highly producing GSH was screened by 96-well plates. Secondary screening was carried out by comparing fermenting power and GSH concentration after strain culture in shaker. The mutant strains Y18-20 GSH after screening by 96-well plates producing 46.8 mg/L GSH in the simulation of cider fermentation conditions, and it’s cell capacity of producing GSH is 8.95 mg/g which is 2.18 times of Y-18. The 96-well plates high-throughput screening method has the advantages of saving the economic costs, improving the filtering efficiency, and shortening the breeding cycle. The mutant strain Y18-20 producing high yield of GSH has better fermentation characteristics and genetic stability, and has a realistic significance to produce high GSH cider, control the browning of cider, and improve the quality of apple wine.