Abstract: To optimize the fermentation conditions of endophytes with antibacterial activity separated from Ficus microphylla, and increase the biomass of fermentation broth and the amount of antibacterial substances, the optimal fermentation condition of the QYPT-B01 strain was optimized by single factor test. The results showed that: the optimum fermentation condition of the QYPT-B01 strain in this experiment was as follows: 3% corn flour, 4% bean cake powder, 0.3% K2HPO4 · 3H2O, initial pH value 8.0, loading amount 30% , fermentation inoculation amount 1%, shaking speed 160 r/min, culture temperature 34℃. Under the condition, the biomass reached the maximum value after culturing 48 h, the OD value of fermentation liquid was 7.95, which increased by 52.0% compared with 5.23 before optimization. The amount of antibacterial substances reached the maximum value after culturing 72 h, the bacteriostasis diameter of fermentation filtrate to Pseudomonas aeruginosa was 26.42 mm, which increased by 13.5% compared with 23.28 mm before optimization. Under the optimized fermentation condition, the biomass of the QYPT-B01 strain and the amount of antibacterial substances increase significantly, which lay a foundation for further separation and purification, identification of antibacterial substances in fermentation filtrate, exploration of the antibacterial mechanism, and the development of antimicrobial agents or new antibiotics.