A pot experiment was conducted to study the effects of biochar combined with different dosages of nitrogen fertilizer on the functional diversity of soil microbial community in flue-cured tobacco rhizosphere. The results showed that biochar application increased the contents of soil organic carbon and microbial biomass carbon, and the treatment of biochar with 15% reduction of nitrogen fertilizer (T3) significantly improved soil catalase activity and metabolism level. Carbon source utilization of soil microorganism were different under different treatments, T3 treatment had the highest average well color development (AWCD) value, and smaller soil microbial richness and dominance. Principle component analysis and thermal graph analysis showed that the functional differences and dominant carbon sources of soil microbial communities were different under different treatments, among of which, T3 treatment had smaller functional difference and contained most carbon sources in the dominant carbon source carboxylic acids. The biochar application improved root-crown ratio of tobacco by 23.06%–42.36%. Therefore, biocar application can enhance the microbial activity of tobacco-planting soil and improve the diversity of soil microbial function, and the biochar combing with 15% reduction of nitrogen fertilizer has the best effect.