Abstract: Bacillus velezensis (TCS001) is a new biocontrol bacterium. The fermentation condition optimization of TCS001 was conducted to improve its biocontrol effect. To obtain the best inhibitory rate of TCS001 fermentation filtrate against pathogenic fungus Botrytis cinerea, six media, including NB, BPY, YT, BP, LB, and SOB medium, were screened. The key factors of fermentation filtrate inhibition rate were investigated using Plackett-Burman (PB) design. Moreover, the central axises of these factors were determined using steepest-ascent method. Optimal fermentation conditions were determined by Box-Behnken design (BBD) and Response Surface Methodology (RSM). Besides, the inhibitory activity of the fermentation broth against B. cinerea was evaluated via artificial spray. The NB medium was found as the best initial fermentation medium for TCS001. And its optimal fermentation parameters were as follows: 10.0 g of peptone, 12.5 g of glucose, 4.0 g of beef extract, 1.0 g of yeast extract , 1 000 mL of distilled water, loading volume of liquid 40%(V/V), inoculum amount 3%(V/V), 164 r/min of shaking speed at 25 °C, initial pH 7.0 , 16 h of strain age, and 36 h of fermentation incubation. The in vitro antibacterial activity test showed that the optimized fermentation broth had the strongest inhibition rate of 90.9%. The disease control experiment showed that the inhibition rate on the disease lesions of B. cinerea was 74.7%, which was 36.9% higher than that before the optimization. The results have provided a theoretical basis for the subsequent research on the separation and extraction of active products and structural identification of TCS001.