Abstract: To investigate the effects of mulberry-soybean intercropping and different nitrogen application rates on extracellular enzyme activity and microbial nutrient limitation in the rhizosphere of soybean, a split-plot design was adopted, with 3 nitrogen levels: no nitrogen (N0), low nitrogen 55.2 kg(N)∙hm⁻², LN, and high nitrogen 82.8 kg(N)∙hm⁻², HN, under monocropping of soybean and soybean-mulberry intercropping. Soil nutrient content and extracellular enzyme activity in the rhizosphere of soybean were measured to assess the impacts of treatments on microbial nutrient limitation. Under intercropping, soil organic carbon, available nitrogen, and soil available phosphorus contents are higher than those in monocropping under N0, with 16.9% and 8.6% increase for available nitrogen and soil available phosphorus, while pH is significantly reduced. In the monoculture system, compared with the N0 treatment, the available nitrogen content in the LN and HN treatments increases by 18.1% and 32.1%, while the soil available phosphorus content decreases by 18.0% and 30.7%, respectively. Intercropping significantly increases the activities of β-glucosidase (BG), acid phosphatase (AP), and N-acetylglucosaminidase (NAG), but significantly decreases vector length and vector angle, which effectively alleviates microbial carbon and phosphorus limitations. BG, NAG, AP activity, and vector angle all increase with the increase of nitrogen application rate, the vector angle of LN treatment is close to 45°, which most effectively alleviates microbial nitrogen limitation without increasing phosphorus limitation. Leucine aminopeptidase (LAP) activity decreases with increased nitrogen levels. Soil available nutrients, pH, microbial biomass, and their stoichiometric ratios significantly affect extracellular enzyme activity and its vector characteristics. Soil available phosphorus and MBC: MBN are the major predictors for vector length and vector angle. Nitrogen fertilization has positive effect on vector angle, while soybean-mulberry intercropping has the greatest negative cumulative effect on vector length. Soybean-mulberry intercropping combined with low nitrogen application 55.2 kg(N)∙hm⁻² could improve soil nutrient availability and extracellular enzyme activity to alleviate microbial nutrient limitation.