Abstract: To obtain antagonistic biocontrol bacteria for tobacco bacterial wilt control, a strain (Xe01) with strong antibacterial activity against Ralstonia solanacearum was isolated from the roots of healthy tobacco plants in Yongzhou, Hunan Province. It was found that the diameter of inhibition zone was 30.5 mm by confrontation culture method, and the strain was identified as Bacillus amyloliquefaciens by morphological observation, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The control effect of antagonistic bacterium Xe01 on tobacco bacterial wilt was 56.32% in greenhouse and 61.46% in plot test. The optimum medium for the antagonistic strain Xe01 was glucose 9.0 g/L, beef extract 3.0 g/L, calcium chloride 4.0 g/L, and the optimum fermentation conditions were pH 7.0, 30℃, 180 r/min, culture 52 h. Under optimized fermentation conditions, the diameter of inhibition zone increased from 30.5 mm to 42.5 mm, increased by 39.34%. The results from this study provided a theoretical basis for the development and application of biocontrol agent using Bacillus amyloliquefaciens Xe01.