Abstract: The effect of bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt on soil microbes and the disease resistance were researched by field experiment. The results revealed that the incidence of tobacco bacterial wilt in the treatment 3 and 4 of adding the bio-organic fertilizer decreased evidently. Compared with the local conventional fertilization, the incidence of the disease decreased by 21.9%, 25.0% in T3 and T4, respectively, while the yield of fluecured tobacco and the proportion of mid-high grade tobacco leaves increased by 5.7%, 5.3% and 2.3%, 2.6%, respectively. The amount and species of rhizosphere microorganisms were much more. While the amount of soil bacteria observably increased by 218.5% with less species once the tobacco bacterial wilt occurred. It could conclude that the bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt could improve the ecological condition to inhibit the growing of pathogenic bacteria to decrease the occurrence and improve tobacco quality.