To prevent and control tobacco bacterial wilt disease with antagonistic bacterium against Ralstonia solanacearum, the microbial diversity in soil of tobacco bacterial wilt disease nursery was preliminarily analyzed by means of conservative fragment amplification, construction of library and bio-information comparison. The results indicated that there existed abundant microbial communities including not only Burkholderia fungorum, Pseudomonas graminis, Aspergillus niger and Fusarium oxysporum, but also some uncultivable bacterium in the soil. The analysis of microbial diversity in the soil could provide some information about microbial community structure for the study on colonization and proliferation process in soil in the application of antagonistic bacterium against Ralstonia solanacearum.