Abstract: To obtain effective biocontrol agents against tobacco black shank and enhance control efficacy, plate confrontation assays were used to screen antagonistic bacteria from tobacco growing soil, and antagonistic strains were identified based on morphological, physiological and biochemical test and phylogenetic analysis. The fermentation medium formula was optimized using a single-factor experimental design, and the control efficacy of the combination of antagonistic bacterial fermentation broth and the chemical agent fluopimomide against black shank was determined through pot and field experiments. The results showed that the screened antagonistic strain GM24 was Bacillus amyloliquefaciens exhibiting an inhibition rate of 60.9% against Phytophthora nicotianae and strong inhibitory activities against various pathogens of tobacco diseases. Through plate confrontation assay combined with microscopic observation, GM24 caused disordered branches, terminal swelling and deformation of pathogen hyphae, thereby inhibiting pathogen growth. The optimal fermentation medium for GM24 was YSP medium, with sucrose, peptone and MgCl₂ as the preferred carbon source, nitrogen source and inorganic salt, respectively. In the pot experiment, the control efficacy of GM24 fermentation broth in combination with fluopimomide at a volume ratio of 5:1 against Phytophthora nicotianae was 82.1%, while in the field experiment, the control efficacy was 67.3% against tobacco black shank, significantly higher than that of the single-agent treatments.