Abstract: The fermentation conditions of the antagonistic bacteria 12-47 to cotton wilt were studied by using the single-factor method. The main factors affecting fermentation conditions include C and N sources, inorganic salts, pH, temperature, rotation speed. The best fermentation condition of each factor were obtained as follows: C source fructose, N source casein protein, inorganic salts MnSO4 and K2HPO4, pH8.0, inoculation volume 8%, seed culture time 72h.The concentration of fermentation conditions were optimized through orthogonal design and the final fermentation medium have more fermentation productions compared with some other common medium. The final optimization of the medium: fructose 3%, casein protein 5%, 0.1% Mn2+, K+ 0.05%, pH 8.0, shaking flask volume is at 100mL/250mL, Inoculation volume 8%, the time of fermentation is 48 hours. Antibacterial activity raise 269% after fermentation than before.