The strains having antagonistic activity against Verticillium dahliae Kleb were isolated and screened from the soil of cotton fields from different regions. A strain named LZ2-70 with a rather higher antagonistic activity was obtained. The morphology characteristics, physiological and biochemical properties and 16S rRNA gene sequence of this strain were further studied. The strain LZ2-70 was finally identified as a type of Bacillus subtilis. The antagonistic substance was antifungal protein. Through single factor experiment and orthogonal experiment, the optimal shaking flask fermentation condition of strain LZ2-70 was determined as follows: medium composed of 5% amidulin, 1% peptone, 0.03% MnCl₂, 0.01% KCl, initial pH 8.0 and 10% inoculation volume, media volume 30/250 (mL/mL), fermentation temperature 37°C, rotating speed 200 r/min, fermentation time 96 h. A distinguished elevation of the antagonistic activity was observed at about 41.1%.