Abstract: Five treatments were analyzed to investigate the changes in soil N fractions and their relationship with soil chemical properties, enzyme activities and gene abundance in different fertilization treatments, including no fertilization (CK), single application of inorganic fertilizer (NPK), single application of organic fertilizer (OM), combined application of inorganic fertilizer and organic fertilizer (NPKO), and combined application of inorganic fertilizer and winter green manure (NPKG). The results showed that:(1) Compared with CK and NPK, the OM and NPKO treatments significantly increased the contents of soil total nitrogen, microbial biomass nitrogen, water-soluble organic nitrogen and nitrate nitrogen. (2) Compared with CK, the OM and NPKO treatments significantly increased whole carbon, microbial biomass carbon and effective phosphorus contents of the soil; Compared with NPK, the OM treatment had a significant effect on carbon composition and available phosphorus contents. (3) Compared with CK, the OM, NPKO and NPKG treatments significantly increased soil ammonia monooxygenase, nitrate reductase and urease activities and functional gene abundance of amoB, nirK, nirS and nosZ by 9.47%~31.13%, 46.46%~1613.48%, 50.41%~323.72%, 108.19%~611.02%, 64.19%~252.15%, 153.20%~373.21% and 56.90%~299.47%, respectively, while NPK significantly reduced the activities of soil nitrate reductase, urease and leucine aminopeptidase and amoA and nirK functional gene abundance. (4) Soil nitrate reductase, urease and nitrous oxide reductase were the main influencing factors for the change of soil nitrogen composition, and there was a highly significant correlation between soil nitrate reductase and soil N fractions (p<0.01). Under long-term fertilization conditions, the activities of key enzymes related to soil nitrogen conversion was a key factor affecting nitrogen conversion in tobacco-planting soils.