Abstract: In this study, Lactobacillus plantarum ZU018 was the research object, the final number of viable cells was as the main reference index, and its fermentation medium was optimized. The single factor experiment was used to select the type and concentration of carbon source and nitrogen source. Maltose, yeast extract and triammonium citrate as the main factors of the medium were preliminarily determined by partial factor test design. The path of steepest ascent and Box-Behnken test design were used to optimize the composition of the medium.The results showed that the optimal optimized medium formula was:Maltose 30.03 g/L, yeast extract 37.50 g/L, beef extract 25.00 g/L, triammonium citrate 4.39 g/L, potassium hydrogen phosphate anhydrous 2.00 g/L, sodium acetate anhydrous 5.00 g/L, magnesium sulphate 0.20 g/L, manganese sulfate 0.05 g/L, and 1.00 g/L tween 80. Compared with that before optimization, the number of viable bacteria in the final fermentation broth of Lactobacillus plantarum ZU018 increased by 4.86 times reaching 10.67×10⁹ CFU/mL, which would provide a theoretical basis for the subsequent high-density fermentation and application of Lactobacillus plantarum.