Biological characteristics of tobacco soil are very important for soil fertility and soil nutrient cycling，and are important biological indexes for evaluating soil fertility. In this paper，the effects of combined application of biochar-based fertilizer on nutrient，enzyme activity and fungal community composition of tobacco-planting soil in Qinling tobacco-growing area under nitrogen reduction conditions were investigated，in order to provide theoretical basis for the efficient utilization of soil nutrients and the improvement of rhizosphere microbial flora structure. A randomized block experimental design was adopted，and two experimental treatments were set up：conventional fertilization（CK），and 750 kg·hm^-2 biochar-based fertilizer + nitrogen reduction by 10%（ST）. Soil samples were collected 75 days after tobacco seedling transplantation，and the rhizosphere soil fungal community and environmental factors were analyzed by high-throughput sequencing technology. Biochar based fertilizer improved the basic physical and chemical properties of soil，increased the α diversity of fungal community，and changed the structure of fungal community. Specifically，biochar-based fertilizers reduced the relative abundance of dominant phyla such as Ascomycota，Basidiomycota and Mucoromycota，and the relative abundance of Blastocladiomycota and Fusarium was increased，while the relative abundance of pathotrophic fungi in rhizosphere of tobacco-growing soil was decreased，which had a certain resistance to plant diseases in the process of plant growth and development. The change of rhizosphere soil fungal community structure was the result of multi-factor interaction. Correlation analysis showed that fungi at phylum level were mostly positively correlated with soil physicochemical properties. The results showed that the application of biochar base fertilizer was beneficial to soil nutrient retention，regulating rhizosphere microbial community structure，increasing the relative abundance of dominant microorganisms，reducing the relative abundance of pathophysiological fungi，and enhancing the resistance of tobacco plants.