Abstract: This experiment aimed to improve the concentration of Lactobacillus plantarum Zhang-LL in the fermentation broth by optimizing culture conditions, which could lay the foundation for obtaining high survival counts of probiotic products. The single factor experiments were carried out to determinate the optimal nitrogen sources of fermentation medium, and rapeseed peptone (501) was the alternative nitrogen source in the MRS culture medium. Then, the single factor experiments and orthogonal experiment L9(34) were employed to optimize fermentation conditions based on the optimized medium. The optimum fermentation conditions were determined as follows: the content of nitrogen source was 3.0%, temperature 34℃, pH 6.5, inoculation 7%, stirring speed 150 r/min, and the pH value controlled at 6.5 by chemical neutralization with 15% Na2CO3. The highest bacterial counts of Lactobacillus plantarum was 1.07×1010 CFU/mL on the optimal conditions.