Abstract: In order to develop highly effective and less toxic bio-control agent for the control of apple tree canker in vitro, the effect of surfactants, preservatives, UV-protectors and carriers on the growing strains of Bacillus amyloliquefaciens TS-1203, and the antibacterial activity of the B. amyloliquefaciens TS-1203 against Valsa mali were determined by the method of mycelium growth rate and response surface methodology(RSM). The results showed that Tween-80 was the most favorable surfactant for the bio-control agent at 0.50% mass fraction. Under this condition, the inhibition rate of TS-1203 was 88.90% at 48 h, which was not significantly different with that of the control. The sodium citrate was the most favorable preservative for the bio-control agent at 0.10% mass fraction. Under this condition, the highest inhibition rate of TS-1203 was 93.39% at 48 h, which was significantly higher than that of the control. Moreover, the sodium lignosulphonate was the most favorable ultraviolet protectant for the bio-control agent at 0.10% mass fraction. Under this condition, the inhibition rate of TS-1203 was 84.18% at 48 h, which was significantly higher than that of the control. The optimum adjuvant for the bio-control agent of TS-1203 was optimized by RSM, and the optimum adjuvant for 65 mL bacterial fermentation contained 650 μL of tween-80, 164.58 mg of sodium citrate and 308.12 mg of sodium lignosulfonate. The inhibitory rate of TS-1203 formulation against V. mali as high as 84.78%, under these conditions. In addition, the diatomite had the strongest effect on the growth of TS-1203, but it was easy to be layered. Sodium alginate had the strongest inhibitory effect on the growth of TS-1203. Therefore, the optimum carrier for bio-control agent TS-1203 was organic bentonite.