Abstract: To prevent and control mildew on cigar tobacco leaves in their fermentation process by biocontrol agents, an antagonistic strain with better mildew inhibitory effect was isolated from non-mildewed cigar tobacco leaves using the plate confrontation method. The fermentation conditions for the isolated antagonistic strain were optimized using a combination of single-factor experiments and response surface methodology. A biocontrol bacterial agent was prepared using vacuum freeze-drying technology, and its efficacy in preventing mould and its impact on the sensory quality of cigars were evaluated. The results showed that a biocontrol strain LK6 capable of inhibiting Aspergillus flavus was screened and identified as Bacillus amyloliquefaciens. The optimized fermentation conditions for strain LK6 were pH 7, 30 ℃, rotation speed of 194 r/min, and inoculation volume of 6.5%. The freeze-dried bacterial agent was prepared with 10% sucrose as cryoprotectant, and the strain survival rate reached 82.61%. The freeze-dried agent of LK6 could effectively inhibit the growth of A. flavus even at the dilution ratio of 10⁴, and there was no negative effect on the sensory quality of cigars by spraying the agent on cigar tobacco leaves. The stability and application efficacy of the freeze-dried agent of LK6 were satisfactory, providing a basis for using anti-mildew bioagents for cigar tobacco leaves.