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 maize with chemical fertilizers and soybean with chemical fertilizers at conventional rate (CF), maize with chemical fertilizers at conventional rate and soybean with half-rate chemical fertilizers (1/2CF), maize with chemical fertilizers at conventional rate and soybean with organic fertilizers (DM), and maize with chemical fertilizers at conventional rate and soybean without chemical fertilizers (NOF) in a rotational maize-soybean system. Our analysis focused on the characteristics of enzyme activity changes associated with soil carbon, nitrogen, and phosphorus transformation. Compared to NOF treatment, CF and DM treatments 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 14.40%–20.87% during the seed filling stage and 28.30% to 34.83% during the mature stage under DM treatment. 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 0.75 units at the mature stages, respectively compared to NOF. Compared to NOF, the CF and 1/2CF treatments significantly increased soil sucrase (SC) activity, however, the trends for leucine aminopeptidase and β-1,4-N-acetyl-glucosaminidase activities among the three treatments were not fully consistent with that of SC activity. The DM treatment generally enhanced soil enzyme activities (except for β-1,4-N-acetylglucosaminidase at the maturity stage), which can be attributed to the abundant organic carbon sources and nutrients provided by organic manure. Additionally, soil β-1,4-glucosidase (BG) and SC activities under DM treatment were higher than that under NOF treatment, and this trend was consistent with the changes in soil TC, TN, and TP. The results of correlation analysis showed that there were significant positive correlations among the activities of BG and SC and soil TC, TN, and TP. The response of soil microbial nutrient limitation to different fertilizer application treatments indicated that Mollisols microbial nutrient acquisition is in an imbalanced state. CF treatment was limited by C and P, while DM treatment was only P limited at seed filling and mature stages. Long-term fertilizer application significantly altered the physicochemical properties and stoichiometric characteristics of Mollisols, with organic manure application showing the most significant effects on enhancing soil nutrient content and improving soil enzyme activities. Moreover, organic manure application was more effective in alleviating phosphorus limitation compared to chemical fertilizer application, providing theoretical support for the conservation of Mollisols and sustainable agriculture.