To study the effects of the combined application of tobacco stem biochar and chemical fertilizers on soil organic carbon fractions and soil microorganisms, a pot experiment was conducted with four fertilization levels, including blank control (CK), chemical fertilizers (F), tobacco stem biochar (B), and tobacco stem biochar combined with chemical fertilizers (BF), and the combined application of chemical fertilizers according to the mixture of the chemical fertilizers (N:P₂O₅:K₂O=12:7:22) with tobacco stem biochar. The results show that, 1) compared to B treatment, soil pH is decreased by 6.59% under BF. Also, BF results in a significant increase in the contents of soil organic carbon (SOC) and easily oxidized organic carbon (EOC), while soil soluble organic carbon content is decreased significantly by 29.63%. However, the effects of BF on extremely unstable soil particulate organic carbon (POC) and microbial biomass carbon (MBC) are not significant. 2)Soil dehydrogenase activity is significantly increased nearly twice under BF compared to F, while soil invertase, dehydrogenase and catalase activities are decreased under BF compared to B. 3) BF improves the richness and species diversity of soil bacterial community, Compared with B, the relative abundance of soil Chloroflexi which is involved in soil organic matter decomposition is increased by 16.79%, while the α and β diversities of soil bacteria are decreased. 4)RDA analysis shows that SOC, EOC, POC and MBC are the main factors affecting the decomposition of soil organic matter and the distribution of bacteria involved in carbon cycling. In conclusion, The combined application of tobacco stem biochar and chemical fertilizers can improve SOC, POC, EOC and MBC contents, reduce DOC content, improve soil enzyme activities and bacterial diversities, increase the relative abundances of some bacteria mainly involved in carbon cycle, such as Chloroflexi and Gemmatimonadetes, promote organic carbon cycling, improve soil quality, optimize soil ecological environment, thus can provide reference for tobacco stem waste utilization and tobacco planting fertilization management.