In this study，field experiment was conducted using a bio-organic fertilizer to prevent the tobacco bacterial wiltin Shaowuregion，and the effect of the bio-fertilizer on root rhizosphere soil microbial diversity and the relationship withbacterial wilt control were analyzed though high-throughput metagenomic sequencing method.The results showed thatthe disease index of fertilization treatment was lower than that of control during the three observation periods，the diseaseindex decreased by 31.43% at 95 d after transplantation，and the economic characteristics of the high quality tobacco and the final yield were higher than those of the control. Sequencing results showed that bio-fertilizer application improved theroot rhizosphere soil microbial diversity of tobaccoplant(Shannon indexincreased).Soil bacterial and fungal flora of eachsample under different treatments were mostly thesame，but the relative abundance wasdifferent，which was related to the occurrence of bacterial wilt.