Abstract: The black soil region of Northeast China, one of the world’s four major chernozem areas, is crucial for national food security. However, long-term reliance on chemical fertilizers has led to nutrient imbalances, soil degradation, and declining microbial diversity, highlighting the need for sustainable fertilization practices. The integration of organic fertilizer with chemical fertilizers has been recognized as an effective strategy for improving soil fertility and maintaining ecological functions. This study analyzed the effects of different fertilization treatments — no fertilizer (CK), chemical fertilizer (NPK), and three rates of organic fertlizer combined with chemical fertilizers (NPKO1, NPKO2, and NPKO3) — on soybean rhizosphere soil nutrient stoichiometry, basal respiration, and microbial community structure in a 21-year field experiment conducted at the Hailun Agroecosystem National Observation Station. Compared to NPK treatment, organic fertilizer treatments significantly reduced C/N while increasing C/P and N/P. Cumulative emissions of CO₂ increased with higher organic fertilizer application rate, with NPKO3 reaching 154.4 mg·kg⁻¹ — 32.86% and 16.07% higher than NPKO1 and NPKO2 treatments, respectively. The combined application of organic and chemical fertilizers enhanced microbial diversity, with the bacterial and fungal Shannon indices under NPKO3 treatment being 12.46% and 15.83% higher than those under NPK treatment. Dominant bacterial phyla included Proteobacteria, Firmicutes, and Actinobacteriota, while fungal communities were dominated by Ascomycota and Basidiomycota. LEfSe analysis identified the highest number of bacterial biomarkers under NPKO3 treatment (66 taxa) and fungal biomarkers under NPK treatment (17 taxa). Principal coordinate analysis (PCoA) revealed distinct shifts in bacterial and fungal community structures across treatments. Partial least-squares path model further indicated that soil nutrient stoichiometry directly influenced microbial diversity, which was positively correlated with cumulative emissions of CO₂. In conclusion, the long-term integrated use of organic and chemical fertilizers improved rhizosphere nutrient balance, microbial diversity, and basal soil respiration in soybean systems. These findings underscore the importance of organic amendments in regulating soil–microbe interactions, providing a scientific basis for optimizing fertilization strategies to sustain soil health and promote sustainable agricultural production.