Abstract: In order to screen the antagonistic bacteria strains with biocontrol effects against tobacco brown spot disease, taking Alteraria alternata as the target bacteria, the soil samples were collected by a 5-point sampling method from the rhizospheric soil of healthy tobacco plants in fields with serious tobacco brown spot disease. A bacterium strain, L249, against Alteraria alternata with an inhibition rate of 72.27% was screened by the gradient dilution isolation method and plate confrontation method. The strain L249 was identified as Bacillus subtilis through colony morphological, physiological, biochemical, and molecular biological tests and could produce biocontrol-related enzymes such as protease, cellulose, and nitrate reductase. The growth rate of strain L249 was measured by turbidimetry, and the results showed that the bacteria entered the logarithmic growth phase after 4 h and entered the stable growth phase after 14 h. The results of pot experiments revealed that fermentation broth of strain L249 could effectively inhibit the occurrence of tobacco brown spot disease with a relative control efficiency of 60.66%, which was not significantly different from that of 10% difenoconazole WDG. Thus, Bacillus subtilis strain L249 can effectively control tobacco brown spot disease and has a good potential for biological control.