[Background] Banana wilt is a fungal devastating soil-borne disease caused by Fusarium oxysporum f. sp. cubense (Foc). In recent years, the application of biocontrol bacterium is an effective means of prevention and control. [Objective] Biocontrol bacteria with good control effect were isolated and screened from the rhizosphere soil of banana, and the number of biocontrol bacteria and antibacterial efficiency were improved by optimizing the medium and fermentation conditions. [Methods] The rhizosphere soil was collected from orchard in Zhangzhou, Fujian province. The pathogen of Foc Tropical Race 4 (Foc 4) was used as indicator fungus. An antagonistic strain named HQB-1 with strong antifungal activity was isolated by dilution coating and plate confrontation method. Strain HQB-1 was identified by morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis. The fermentation conditions were optimized by single factor experiment and orthogonal experiment design. [Results] Strain HQB-1 was identified as Burkholderia stagnalis, the optimized medium was beef extract 5.0 g/L, yeast extract 10.0 g/L, NaCl 5.0 g/L, and the optimal culture temperature at 27 °C, pH 7.0, rotation speed 200 r/min, inoculation amount 1%, and the culture time 36?h. [Conclusion] The number of effective viable cells and the inhibition rate were, improved significantly than before. The OD600 was increased from 1.251 to 1.881, and the inhibition rate was from 9.18% to 34.60%.