Abstract: To effectively control the mildew of stored tobacco leaf, a Bacillus strain LL17 was isolated from tobacco rhizosphere soil. A dual culture method and Oxford cup method were used to study the antagonistic effects of strain LL17 on three kinds of mildew fungi. The bacteria cultural, morphological and biochemical features were observed, and in addition 16S rDNA sequence analysis was used to identify the strain. The results showed that strain LL17 was identified as Bacillus subtilis on the taxonomy. The inhibition zones were clearly observed in PDA by using the dual culture method and Oxford cup method, indicated that strain LL17 was strongly antagonistic towards three kinds of mildew fungi. Sterile filtrate of strain LL17 was antagonistic towards 2 kinds of Aspergillus conidiospores. The sterile filtrate could affect spore germination or induce hyphae deformation. Therefore, strain LL17 might have the potential application prospect and value in biological control of tobacco mildew during storage.