Abstract: In order to clarify the biocontrol characteristics of Bacillus velezensis GY1, Bacillus amyloliquefaciens GY10, and Bacillus subtilis GY12, antibacterial spectrum, secondary metabolites, volatile substances, rhizosphere colonization ability and growth-promoting ability of the strains were studied by compatible test, double petri dish assay and pot test. The results showed that GY1, GY10, and GY12 can significantly promote seed germination and growth of tobacco. The three strains have a broad antimicrobial spectrum and have significant inhibitory effects against Alternaria alternata, Phytophthora parasitica var. nicotianae and Fusarium spp. In addition, all of the tree strains have the ability to produce siderophore and protease. The fermentation supernatants and volatile substances of the three strains also have a significant inhibitory effect against A. alternata and P. parasitica var. Nicotianae. The secreted proteins of the strains have good thermal stability and significant in vitro antibacterial activity on A. alternata, and have high tolerance to acid environments. The results of rhizosphere colonization assay indicated that GY1-GY10-GY12 compounding could increase the colonization of each biocontrol bacteria in the tobacco rhizosphere compared with a single strain. The resuls from this study provide guidance for in-depth exploration of the biocontrol characteristics of strains GY1, GY10, and GY12 and the industrial development of biocontrol strains.