Abstract: In order to develop an effective biocontrol agent against apple canker, which was caused by the pathogen of Cytospora spp., the antagonistic bacteria strains were isolated, screened and identified by the method of confronting incubation, morphology and molecular biology. In addition, the control effect of antagonistic bacteria strains against Cytospora spp. in detached apple twigs was determined in vitro, and the probable mechanism and antibacterial activity of antagonistic strains against Cytospora spp. conidium germination and hypha growth were determined by microscopic observation and liquid culture. Twenty-three strains of bacteria were isolated from the apple orchard soil and apple tree branches in apple production areas of Gansu Province. And two antagonistic bacteria strains of LZ-1201 and TS-1203 showed significantly antagonistic effect on the Cytospora spp. growth. The inhibition rates of LZ-1201 and TS-1203 against Cytospora spp. hypha growth were 79.00% and 85.00%, respectively. The strains of LZ-1201 and TS-1203 were identified as Bacillus subtilis and Bacillus amyloliquefaciens, respectively. The results of control experiments showed that the control effect of two antagonistic bacteria strains against Cytospora spp. decreased with the increasing dilution multiple of fermentation filtrate. The undiluted fermentation filtrates of LZ-1201 and TS 1203 strains had the best control efficiencies, which were 74.43% and 77.07%, respectively. Analysis of the probable antifungal mechanism of antagonistic bacteria strains indicated that two antagonistic bacteria strains could induce the mycelium of Cytospora spp. enlargement and deformity, cytoplasm exosmosis and dissolution. The fermentation filtrate of two antagonistic bacteria strains significantly reduced the conidium germination rate and hypha growth. When diluted by 40 times, the inhibition rate of fermentation filtrates of antagonistic bacteria strains on hypha growth was more than 60%. Therefore, these results suggested that the antagonistic strains provide a new biological control method in controlling apple canker.