Currently, China’s soybean self-sufficiency rate is only 15%, highlighting the soybean crisis and the supply chain risks that pose a major threat to China’s food security. Thus, it has become imperative to step up efforts to boost soybean production capacity while promoting the green and sustainable development of regional farmland ecosystems. In this context, the present study comprehensively investigated the effects of intercropping and nitrogen application rate on soybean yield, as well as the changes in gradients generated by different levels of nitrogen application. Based on six consecutive years of maize–soybean intercropping planting patterns, the inter-root soils of soybeans were collected at the flowering stage and evaluated for soil nitrogen content, nitrogen-assimilating enzyme activities, and microbial community composition of soybean, which were correlated with yield, to clarify the main pathways and modes of intercropping effects. The N2 level (80 kg·ha−1) was favourable for higher yield. In comparison to monocropping, the intercropping reduced yield by 9.65–13.01%, photosynthetic characteristics by 1.33–7.31%, and plant nitrogen-assimilating enzyme activities by 8.08–32.01% at the same level of N application. Likewise, soil urease and catalase activities were reduced by 9.22 and 1.80%, while soil nitrogen content declined by an average of 6.38%. Gemmatimonas and Bradyrhizobium enrichment significantly increased soil nitrogen content, photosynthetic characteristics, and soybean yield, while it was reduced by Candidatus_Udaeobacter and Candidatus_Solibacte enrichment. The results of this study provide a theoretical basis for further optimising maize–soybean intercropping, which is crucial for enhancing the agricultural production structure and improving the overall soybean production capacity.