Abstract: The research aims to provide a theoretical basis for the reasonable application of Bacillus. amyloliquefaciens. Field experiments and ITS amplicon sequencing were conducted to explore the effects of three application methods of B. amyloliquefaciens on improving the fungal community in tobacco rhizosphere soil. The three application methods included the followings: (1)diluted with water and then irrigated into tobacco rhizosphere (DS), (2)mixed with soil and then applied around tobacco rhizosphere (BT), (3) mixed with rice bran and soil, then applied around tobacco rhizosphere (MK+BT). Results showed that the treatment of BT and MK+BT could significantly improve the OTU number and the Sobs, Shannon, Chao 1 indexes of soil fungal community. Meanwhile, BT and MK+BT can also significantly promote the structure of soil fungal community, improve the relative abundance of fungal phyla (Ascomycota, Basidiomycota), and increase the proportion of beneficial fungal genera (Chaetomium, Trichoderma, Gibberella). In conclusion, the promotion tendency of three application methods of B. amyloliquefaciens on fungal community in tobacco rhizosphere soil was shown as MK+BT > BT > DS.