Abstract: This experiment aimed to improve the concentration of bacteria in Bacillus 13002 fermentation medium by improving the culture conditions, and to lay the foundation for the production of high optical purity L-lactic acid. The single factor test, steep climbing test and Box-Behnken response surface test were used to confirm the optimum culture medium and optimum culture conditions of Bacillus 13002 and high density batch feeding was carried out in automatic fermentation tank. Results showed that the optimal medium was glucose 20 g/L, bacterial peptone 10 g/L, yeast extract powder 25 g/L, Na Cl 10 g/L, initial p H6.5.Under the optimum condition, when 6% Bacillus 13002 and nutrients were added at the rate of 0.5 g/ ( L·h) from the 16 th hour to the terminal at 45 ℃, the maximal cell concentration was 5.399 g/L.The total number of bacteria was3.095 × 10⁹ CFU/m L, and reached the level of 0.730 × 10¹¹ CFU/g after lyophilization.