Abstract: This study aims to investigate the effect of long–term fertilizer application on microbial nutrient limitation in Mollisols through ecological stoichiometry and enzyme metrology. The study was conducted at the Monitoring and Research Station of Black Soil Erosion, affiliated with the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences. We compared the effects of normal chemical fertilizer application (CF), reduced chemical fertilizer application (1/2CF), substitution of chemical fertilizer with organic manure (DM) and no fertilization (NOF) in a rotational corn–soybean system. Our analysis focused on the characteristics of enzyme activity changes associated with soil carbon, nitrogen, and phosphorus transformation. Compared to no fertilization (NOF), the application of chemical and organic fertilizers substantially increased the total carbon (TC), total nitrogen (TN), and total phosphorus (TP) content in Mollisols during both the seed filling and mature stages. Specifically, TC, TN and TP levels rose by 14.4%–20.9% during the seed filling stage and 28.3% to 34.8% during the mature stage. Organic manure application had the most substantial effect on enhancing soil nutrient content levels. Furthermore, soil pH in DM treatment increased significantly, rising by 0.69 units at the seed filling stage and 0.82 units at the mature stages, respectively compared to NOF. Compared to NOF, the CF and 1/2CF treatments significantly increased soil sucrase (SC) activity, whereas nitrogen-decomposition enzyme activities (LAP, NAG, and LAP+NAG) exhibited a decreasing trend. The DM treatment generally enhanced soil enzyme activities, which can be attributed to the abundant organic carbon sources and nutrients provided by organic manure. Additionally, the activities of β-glucosidase (BG) and SC at different stages were consistent with the trends of soil TC, TN, and TP changes, and showed significant positive correlations. The response of soil microbial nutrient limitation to different fertilizer application treatments indicated that Mollisols microbial nutrient acquisition is in an imbalanced state. Compared to NOF, chemical fertilizer application was limited by C and P, while organic manure application was only P limited at seed filling and mature stages.