Abstract: In order to screen strains of antagonistic bacteria against cucumber fusarium wilt, one bacteria strain was isolated from the rhizosphere soil of cucumber by the methods of plate confrontation, and the inhibitive efficiency on the hyphal growth of Fusarium oxysporum f. sp. cucumeris was tested. The results showed that the antagonistic bacteria strains Th-1 had significant inhibition effect on Fusarium oxysporum f. sp. cucumeris. By the homology analysis of 16S rRNA sequence together with morphological, physical and biochemical characteristics, Th- 1was identified as Bacillus subtilis and its fermentation conditions was optimized. The optimal fermentation conditions were composed of the following factors: seed age, 10 h; inoculum, 4% (v/v); fermentation temperature, 32?C; liquid volume, 50 mL/250 mL; rotation speed, 180 r/min; incubation time, 36 h. This optimization strategy enhanced the fermentation liquid colonies nearly 1.72 times of the initial value.