The high density fermentation technology of Lactobacillus R8 strain was studied in this study. The main reference indexes were the density (OD600) and the utilization of carbon source (glucose). The effects of medium and agent, pH value, inoculation amount, initial sugar content, aeration mode and feeding process on the strains growth in 7.5 L stirred fermentation tank were studied, and then the experiment bacteria were optimized by 250 L pilot-scale amplification. The optimizing small-scale fermentation process of the bacteria was: inoculating the seed liquid (8% v/v) into cannikin with medium volume of 70%, , the initial sugar concentration 40 g/L, stirring speed 100 r/min, intermittent nitrogen to maintain a certain anaerobic environment, automatic flow plus 12.5% ammonia, and the fermentation at 37 ℃ and pH 5.8. The 250 L pilot fermentation process was as follows: the pH was controlled at 5.8, the fermentation was performed at 37 ℃ for 7 h, the seed liquid was inoculated in the 250 L fermentation tank of 70% liquid at 8% inoculation amount , stirring speed was 60r/min, 12.5% ammonia was used to maintain the pH of medium at 5.8, , intermittent nitrogen was introduced (0.2 vvm/2 h of the rate of nitrogen 5 min), the resultant mixture was incubated for 10 h at 37 ℃. The live bacteria concentration of fermented liquid obtained from 250 L scale was about 8×109 CFU/mL, and the concentration of live bacteria was about 1.2×1011 CFU/g after vacuum freeze-drying.