Abstract: To clarify the effects of Bacillus subtilis BC80-6 on Thielaviopsis basicola, the inhibitory effects of three treatment solutions of B. subtilis BC80-6 on the mycelial growth and spore germination of T. basicola were investigated. BC80-6 fermentation conditions were optimized and field control experiments were carried out. The results showed that 10^-1 times living bacteria solution of B. subtilis BC80-6 had the best inhibitory effects on the mycelial growth and spore germination of B. subtilis with the inhibition rates of 93.2% and 95.3%, respectively. Via a single factor test and an orthogonal experiment, the optimum BC80-6 medium was 1.5% sucrose, 0.5% yeast extract and 0.06% magnesium sulfate (mass/volume ratio). The best fermentation condition was fermentation temperature 28℃, time 24 h, initial pH 6.5, rotation speed 180 r/min, inoculation amount 5% (v/v) and medium volume 40 mL in 250 mL triangular bottle. The field control effect of living bacteria solution of B. subtilis on tobacco root black rot was 88.19%.