Abstract—Edible and medicinal mushrooms are healthy foods relatively low in calories and fat but rich in proteins, vitamins, minerals and ergothioneine. After mushrooms are harvested, they continue to breathe and metabolize. Therefore, many physiologically active contents will change after harvest. Due to the different storage time and cooking ways, we studied a variety of ergothioneine content to determine the content of ergothioneine in Flammulina velutipes, Pleurotus citrinopileatus and Pleurotus eryngii after processing and cold storage. We compared the ergothioneine after different storage days in 4℃, dry way (freeze dry, cold air dry, hot air dry), radiation dose (pulsed light), cooking method (boil, stir-fry, gas fry) with high performance liquid chromatography to determine the process that can keep most ergothioneine. Ergothioneine content decreased while P. citrinopileatus storage days increased, and the highest content of ergothioneine was found to be 2241.6 μg/g on the third day. After irradiation of different dose of pulse light, the content of ergothioneine had no significant difference between 30 and 50 pulse form three types mushroom. The lowest content is to cook in boiled water for 3~10 minutes. Therefore, storage for 3 days, irradiation 50 pulse light, boiling for 3~5 minutes, gas-frying for 6 minutes, stir-frying for 10 minutes, and freeze drying was the best way to keep the ergothioneine in P. citrinopileatus. However, the processing treatment had little effect on F. velutipes and P. eryngii, except for boil treatment. Food processing and storage has the potential to slightly alter the stability of ergothioneine in mushrooms. Consumer guideline for estimating dietary intake of ergothioneine is to not boil mushrooms for too long, in order to retain more ergothioneine content.