Abstract: In order to investigate the effects of biochar on physicochemical properties and microbial community of tobacco-planting soil, 1.5 t/hm² (B1.5) and 15 t/hm² (B15) biochar additions were set with fallow (FL) and single fertilizer (CK) as the control, and the effects of different amounts of biochar on the physical and chemical properties and fungal community of tobacco-planting soil were researched. The results showed that biochar amendment significantly increased soil pH, moisture content, organic matter content and the proportion of ≥0.25mm aggregates, as well as significantly reduced soil bulk density. The mean mass diameter (MWD), microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) contents of aggregates increased with the increase of biochar addition. In general, the physical and chemical properties of soil treated with B15 were better than those treated with B1.5. The addition of biochar had no significant effect on the soil fungal diversity index, but changed the relative abundance of major fungal phyla. The relative abundance of Ascomycota, Mortierellomycota and Mucoromycota of B15 was significantly higher than that of CK. The addition of biochar also increased the relative abundance of Gibberella, Mortierella and Plectosphaerella. The soil fungal community structure was changed by biochar, but it was similar at two biochar amendment levels. The results of redundancy analysis (RDA) and Mantel test showed that soil pH, organic matter, MBN and aggregates (≥0.25 mm) were the main factors affecting the fungal community in tobacco-planting soil. In conclusion, the addition of biochar of 15 t/hm² is more conducive to the comprehensive optimization and sustainable development of soil productivity.