Abstract: Bacillus licheniformis W10 is an antagonistic bacterium, which can produce antifungal protein inhibiting growth to a variety of plant pathogenic fungi. The technical conditions of fermentation W10 to produce antifungal protein were studied. The results indicated that yield of antifungal protein was promising under the conditions of inoculation bacteria for 12 h, 2% inoculum volume, fermentation for 60 h and pH 7.0. During the fermentation, changing culture temperature was favorable to the antifungal protein accumulation and increased its inhibitory effect against plant pathogen Botrytis cinerea, with fermentation temperature of first stage was 30 ℃ for 20 h, and then was adjusted to 28 ℃ until the end of fermentation. The suitable dissolved oxygen value was 20%. The better fermentation method was two fed-batch culture with 10 g·L^-1 glucose and 5 g·L^-1 peptone as the supplement medium. The defoaming substance was defoamer GPE (polyoxypropylene-polyoxyethylene glycerol ether) with 200 mg·L^-1. Under the optimized fermentation conditions, the inhibitory effect of the strain W10 against B. cinerea reached 99.5%, increasing 34.6% compared with the control.