Abstract: In order to reduce the disadvantages of chemical control of Phytophthora nicotianaeand improve the effect of biocontrol agents, Trichoderma asperellum HG1 with obvious anti-Phytophthora nicotianaeactivity was obtained by the agarose diffusion method and the a co-culture system of Trichoderma asperellum HG1 and Bacillus subtilisTpb55 was constructed to evaluate the inhibition effect of interspecific interaction against Phytophthora nicotianae by the mycelial growth rate method. The composition of medium media and fermentation conditions were optimized by single factor experiment. The control effects of co-culture and single-culture fermentation liquors of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55 on tobacco black shank were compared by pot experiments. The suitable co-culture system was determined as follows: the inoculation ratio of HG1 (10⁶ CFU/mL) and Tpb55 (10⁶ CFU/mL) was 10:1. Sequential inoculation was adopted as HG1 was inoculated first, and Tpb55 was inoculated after 24 h. The fermentation broth composition and culture conditions of the co-culture system were optimized by single factor method, guiding by the inhibition rate of Phytophthora nicotianae. The optimum medium composition was determined as follows: xylose 10 g/L, peptone and yeast powder (the mass ratio was 2:1) 5 g/L; The optimal fermentation conditions were as follows: temperature 25 ℃, initial pH 7.5, rotating speed 140 r/min. The disease control effect of co-culture fermentation liquors in pot experiments was 74.72%, which was significantly better than that of single-culture. The system could significantly improve the inhibition activity of the two biocontrol strains against Phytophthora nicotianae, and the fermentation liquors of the system had significant disease control effect on tobacco black shank, which provides a basis for the development of subsequent multi strain co-culture biocontrol agents.