Abstract: Tobacco target spot disease, caused by Rhizoctonia solani, is one of the most important foliar diseases of tobacco. The screening and identification of biocontrol strains, along with the establishment of biocontrol fermentation conditions, can technically support the prevention and control of the disease, and the application of biocontrol bacteria. In this study, a previously screened antagonist strain Y18 was identified through the combination of morphology, physiology, biochemistry and molecular biology. Using the inhibition rate of Y18 on the mycelial growth of the target tobacco blotch as an index, the fermentation medium components and fermentation conditions with the optimal inhibitory effect of Y18 on the target blotch were screened by one-way test and orthogonal test. The strain Y18 was identified as Bacillus velezensis, with the inhibition rate of 81.63% on Rhizoctonia solani. It also had pronounced inhibition on Phytophthora capsici, Fusarium graminearum, Phytophthora parasitica var nicotianae, Ralstonia solanacearum and Pseudomonas syringae pv. tabaci, respectively, with the inhibitory rate reaching 79.08%, and the size of the inhibition circle for Ralstonia solanacearum up to 27.28 mm. The optimal fermentation medium, carbon source, nitrogen source and inorganic salt were NB medium, sucrose, beef paste and magnesium sulphate, respectively. The optimal medium components were 10 g sucrose, 10 g beef paste and 7.5 g magnesium sulphate. The optimal fermentation conditions were pH=6, fermentation time of 48 h, inoculum amount of 5% and fermentation temperature of 26 ℃. The results can provide a theoretical basis for the development and utilization of strain Y18 as a biocontrol agent.