Abstract: To determine the biocontrol effect of Bacillus velezensis F85 against tobacco anthracnose, co-culture experiments of B. velezensis F85 and C. fructicola were conducted. The effects of different concentrations of B. velezensis F85 aseptic fermentation filtrate (0.5%-20%) on mycelium growth, conidial germination and appressorium formation of C. fructicola were determined, and the effect of F85 on live leaf control of C. fructicola was evaluated. The results indicated that: (1) B. velezensis F85 showed good antagonism to C. fructicola, C. kahawae, C. karstii and C. siamense, suggesting that B. velezensis F85 had broad spectrum antifungal property for Colletotrichum spp.. (2) B. velezensis F85 could effectively inhibit the mycelium growth of C. fructicola, and inhibit the germination of conidia and appressorium formation of anthrax. With the increase of the concentration of sterile fermentation filtrate of F85, its inhibitory effect on C. fructicola was more obvious. The dry weight inhibition rate of C. fructicola mycelium and the malformation rate of conidial bud tube were both 100% when the filtrate of F85 was 20%. (3) The control effect of B. velezensis F85 (above 5%) on live leaf tobacco anthracnose was higher than 70%. This study enriched the biocontrol bacteria resources for tobacco anthracnose and provided theoretical basis for biological control of tobacco anthracnose.