Soil CO2 emissions are key components of global carbon cycling. Hence, knowledge of their drivers is important for estimating, and modifying, carbon storage pools. To extend knowledge of these drivers, results of a long‐term (31‐year) experiment in a rice–wheat rotation field in subtropical Central China were analyzed to determine effects on soil CO2 flux (FCO2) of various fertilization practices and related environmental factors including soil organic matter (SOM), microbial biomass carbon (MBC), total nitrogen (TN), total phosphorus (TP), and total potassium (TK) contents; activities of three soil enzymes (acid phosphatase, urease, and catalase); and soil temperature, pH, cation exchange capacity (CEC), bulk density and porosity. The results clearly show that FCO2 was sensitive to changes in soil nutrient conditions under different long‐term fertilization practices. Notably, it was significantly higher in plots receiving organic manure applications than in those receiving only chemical fertilizers (N, NP, and NPK) and unfertilized plots. Correlation analysis showed that FCO2 was significantly correlated with the soil's SOM, TN, TP, and MBC contents, acid phosphatase and urease activities, and several physicochemical soil properties including pH, CEC, bulk density and porosity. However, no significant correlations were observed between FCO2 and either TK content or catalase activity. The findings suggest that at given temperature and soil moisture contents, variations in soil CO2 emissions associated with the fertilization practices are strongly related to SOM, and the nutrient contents, microbial and enzyme activities, and physicochemical properties of the soil.