In order to obtain antagonistic bacteria with inhibitory effects on tobacco Potato Virus Y (PVY), broad-spectrum antibacterial activities and biological functions of highly effective antagonistic bacteria against PVY from tobacco rhizosphere soil were screened and characterized. The fermentation conditions for the strain were optimized by a single-factor test, and the pot control effect of the strain on tobacco PVY was verified. The results showed that 216 strains of antagonistic bacteria were isolated and purified, of which 4 strains had a significant antagonistic activity against PVY, and ZF-10 had the best antagonistic effect with an antagonistic rate of 90.20%. The strain was identified asBacillus velezensisby morphological observation, detection of physiological and biochemical characteristics, and molecular biological analysis. ZF-10 showed inhibitory effects on various pathogenic fungi, and had the ability to produce ammonia, cellulase production, protease production, dissolve inorganic phosphorus, and siderophore production, but no ability to release potassium. The optimal fermentation medium for ZF-10 was soluble starch at 15.0 g/L, yeast extract at 25.0 g/L, magnesium sulfate at 7.5 g/L, and the optimal fermentation conditions were culture temperature at 32 ℃, inoculum at 1%, 250 mL flask volume 60 mL, initial pH at 7.0, fermentation time at 36 h. The results of the pot experiment showed that the relative control effect of ZF-10 fermentation broth on tobacco PVY disease was 61.85%, indicating a satisfactory biocontrol potential.