Abstract: The black soil region of Northeast China, one of the world’s four major Chernozem areas, is vital for national food security. However, prolonged reliance on chemical fertilizers has resulted in nutrient imbalances, soil degradation, and reduced microbial diversity, underscoring the need for sustainable fertilization strategies. Organic fertilizer application, particularly when integrated with chemical fertilizers, is considered an effective approach to enhance soil fertility and sustain ecological functions. In this study, a 21-year field experiment was conducted at the Hailun Agroecosystem National Observation Station to assess the effects of different fertilization regimes — no fertilizer (CK), chemical fertilizer (NPK), and three rates of organic manure combined with NPK (NPKO1, NPKO2, NPKO3) — on soybean rhizosphere soil nutrient stoichiometry, basal respiration, and microbial community structure. Organic fertilizer treatments significantly reduced the C/N ratio while increasing the C/P and N/P ratios compared with NPK. Cumulative CO₂ emissions rose with higher organic fertilizer inputs, with NPKO3 reaching 154.4 mg·kg⁻¹, 32.86% and 16.07% greater than NPKO1 and NPKO2, respectively. The combined use of organic and chemical fertilizers enhanced microbial diversity; under NPKO3, bacterial and fungal Shannon indices were 12.46% and 15.83% higher than under NPK alone. The dominant bacterial phyla were Proteobacteria, Firmicutes, and Actinobacteriota, while Ascomycota and Basidiomycota were the dominant fungal phyla. LEfSe analysis identified the most bacterial biomarkers under NPKO3 (66 taxa) and the most fungal biomarkers under NPKO2 (13 taxa). Principal coordinates analysis demonstrated distinct shifts in both bacterial and fungal community composition across treatments. Partial least squares path modeling further revealed that soil nutrient stoichiometry directly influenced microbial diversity, which was positively associated with cumulative CO₂ emissions. In conclusion, the long-term integrated application of organic manure and chemical fertilizers improved rhizosphere nutrient balance, increased microbial diversity, and promoted basal soil respiration in soybean systems. These findings highlight the critical role of organic amendments in regulating soil–microbe interactions and provide a robust scientific basis for optimizing fertilization strategies to sustain soil health and advance sustainable agricultural production.