The invention relates to a high-density enterococcus faecalis fermentation culture medium and a fermentation process thereof. The fermentation culture medium is prepared from 20-30 g / L of glycerol, 10-15 g / L of soybean meal, 1-3 g / L of ammonium sulfate, 2-3 g / L of potassium hydrogen phosphate, 3-8 g / L of sodium acetate trihydrate, 2-4 g / L of trisodium citrate dihydrate, 0.3-0.8g / L of magnesium sulfate heptahydrate and 0.1-0.3 g / L of manganese sulfate monohydrate. The fermentation process comprises the steps that high dissolved oxygen (25%) is adopted and combined with glycerin to serve as a fermentation carbon source, the pH value of the constant sodium carbonate fermentation liquid is 5.5, the glycerol concentration is controlled to 10 g / L by adding a supplementary material in the fermentation process in a flowing mode, culture is performed for 16 hours, and then the fermentation is completed when OD (600 nm) is greater than 35, wherein the viable count of the enterococcus faecalis contained in the fermentation liquid can be up to 8*1010 CFU / mL or above. In the fermentation mode that the high dissolved oxygen is combined with the glycerin to serve as the carbon source, the lactic acid concentration in the fermentation liquid is reduced, the enterococcus faecalis contained in the fermentation liquid can be up to 8*1010 CFU / mL or above, the biomass separation cost is reduced, a production cycle is shortened, the production cost is reduced, and the production efficiency is improved.