Abstract: Effects of single factors,including extraction method,liquid-solid ratio,ethanol concentration and extraction temperature,on extraction rate of Flammulina velutipes ergothioneine were studied in this manuscript. Based on single factor experiments,the optimum process conditions for the ergothioneine extraction were determined using response surface methodology. The results showed that the different extraction methods could significantly(p<0.05)affect the extraction yield of EGT from Flammulina velutipes,of which the solvent extraction had the higher extraction yield and was considered as convenient and low energy consumption process for ergothioneine;The optimum extraction conditions obtained through response surface methodology were as follows:extraction time of 15 min,ethanol concentration of 62%,liquid-solid ratio of 58:1 (g/mL),extraction temperature of 53 ℃;Verification experiment was carried out under the optimum extraction,and the highest ergothioneine yield was(3.53±0.09) mg/g dry weight. No significant difference was found between observed and estimated values(3.48 mg/g dry weight),suggesting that the optimized process was reliable and valid for Flammulina velutipes ergothioneine extraction.