A strain of Bacillus sp. named HMI-23, which has been identified as a member of Bacillus amyloliquefaciens by 16S rDNA analysis, and exhibited extensive antagonistic ability on Rhizoctonia solani，Sclerotinia sclerotiorum，Ralstonia solanacearum was isolated from tobacco-rice-rape rotation soil. The inhibition ability of HMI-23 fermentation broth supernatant was insensitive to proteinase K, ultraviolet ray and high temperature. Four kinds of lipopeptides synthesis related genes have been detected in the genome of strain HMI-23 by PCR. The lipopeptides crude extract of HMI-23, which may contain several known lipopeptide including iturin A, mycosubtilin B, bacillomycin D, and fengycin B by LC/MS analysis, have strong ability to inhibit plant pathogens, such as Rhizoctonia solani, Sclerotinia sclerotiorum and Ralstonia solanacearum.