The invention relates to a 2-keto-L-gulonic acid fermentation process. Culture medium containing sorbose, corn steep liquor, urea, magnesium sulfate, monopotassium phosphate and light calcium carbonate nutrient substances is added into a fermentation container, mixed bacteria liquid which can convert the sorbose into 2-keto-L-gulonic acid is added, fermentation is carried out for 2 to 12 hours, lysozyme is added, the dosage of the lysozyme in the fermentation broth is 10U / ml to 10000U / ml, culture is continued, the culture temperature is controlled at 28 DEG C to 35 DEG C in the whole fermentation process, the culture pH value is controlled at 5.4 to 8.5, and fermentation is finished until the residual sorbose in the fermentation broth is lower than 1mg / ml. Through the process, the dosage of the lysozyme is reduced, the fermentation period is shortened by 10 to 15 hours, the production efficiency is increased, and the production cost is reduced.