Abstract: This study investigated 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. A rotational maize-soybean system was established under four treatments: 1) maize and soybean with chemical fertilizers at conventional rates (CF); 2) maize with chemical fertilizers at conventional rate, and soybean with half-rate chemical fertilizers (1/2CF); 3) maize with chemical fertilizers at conventional rate, and soybean with organic fertilizers (DM); 4) maize with chemical fertilizers at conventional rate, and soybean without chemical fertilizers (NOF). We analyzed the characteristics of enzyme activity changes associated with soil carbon, nitrogen, and phosphorus transformation during soybean growth period. Compared to NOF treatment, CF and DM treatments increased the total carbon (TC), total nitrogen (TN), and total phosphorus (TP) contents in Mollisols during both the seed filling and mature stages; specifically, TC, TN, and TP contents increased by 14.40%–20.87% during the seed filling stage and by 28.30%–34.83% during the mature stage under DM treatment. Organic fertilizer application had the most substantial effect on enhancing soil nutrient contents. Furthermore, soil pH under DM treatment increased significantly by 0.69 unit during the seed filling stage and by 0.75 unit during the mature stage, respectively, compared to that under NOF treatment. Compared to NOF treatment, 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 (NAG) activities among the three treatments were not fully consistent with that of SC activity. The DM treatment generally enhanced soil enzyme activities (except for NAG during the mature 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 were higher under DM treatment 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 significant positive correlations between the activities of BG and SC with soil TC, TN, and TP contents. 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 during the seed filling stage, and by P during the mature stage; DM treatment was limited only by P during the seed filling and mature stages. Long-term fertilizer application significantly altered the physicochemical properties and stoichiometric characteristics of Mollisols, with organic fertilizer application showing the most significant effects on enhancing soil nutrient contents and improving soil enzyme activities. Moreover, organic fertilizer application was more effective in alleviating P limitation than chemical fertilizer application. The results provide theoretical support for the conservation of Mollisols and sustainable agriculture.