Abstract: To clarify the antagonistic effect of strain FC02 against the pathogen of tobacco brown spot disease (Alternaria alternata), the strain was identified by colonial morphological observation, physiological and biochemical identification and phylogenetic tree analysis. The inhibitory effect of strain FC02 on Alternaria alternata was analyzed by confrontation culture method, and the inhibition rate of the fermentation filtrate on hyphal growth of Alternaria alternata and its antibacterial stability under different treatments of varying temperature, pH value, ultraviolet irradiation time and storage time were determined. The control efficiency of the fermentation broth on tobacco brown spot disease was determined by pot experiments. The results showed that: 1) Strain FC02 was Bacillus velezensis. 2) During confrontation culture, the cell wall of Alternaria alternata was ruptured, the cell membrane was invaginated, some organelles disintegrated, the mycelium shrunk and deformed, and no spore was germinated. The inhibition rate of mycelial growth of Alternaria alternata was 78.70%. 3) The inhibition rate of the fermentation filtrate of strain FC02 on mycelial growth was 81.19%, and it had good antibacterial stability in different treatments of temperature (25-120 ℃), pH value (7-11), ultraviolet irradiation time (5-45 min) and storage time (0-60 d). 4) In the pot experiment, the fermentation broth of strain FC02 (1×10⁷ cfu/mL) showed a control efficiency of 71.40% against tobacco brown spot disease, which was not significantly (P < 0.05) different from the control efficiency (68.35%) by 1×10⁹ cfu/g Bacillus subtilis wettable powder.