Root exudates significantly influence soil organic carbon (SOC) formation and persistence. However, how fertilizer application regulates microbial-mediated processes to link root-derived carbon dynamics with long-term SOC sequestration remains to be elucidated. This study was carried out on loess hill farmland with 26 years of nitrogen (N) and phosphorus (P) fertilization. We examined root exudates, soil C fractions, and microbial functional gene abundance related to C cycling via high-throughput gene chip analysis. The findings revealed that applying N and P fertilizers led to notable boosts in soil organic carbon (SOC), reactive organic carbon (KOC), and recalcitrant organic carbon (ROC), with increases of 33%, 30%, and 39% respectively, compared to the control. Mechanistically, N and P fertilization altered root exudate composition, with organic acids (fatty acids, amino acids, and dicarboxylic acids) comprising over 60% of the total exudates. These changes in exudates promoted the enrichment of C fixation functional genes, particularly aclB and acsA, which are responsible for enhancing C fixation in the soil. Additionally, reactive C fractions are positively linked to C fixation gene abundance and negatively associated with C degradation genes, suggesting that fertilization not only boosts C fixation but also limits C loss through microbial degradation. Overall, our findings highlight that N and P fertilization stimulates SOC accumulation primarily by modulating microbial C fixation processes via root exudate composition, offering valuable insights into the microbial mechanisms underlying SOC sequestration in low-fertility farmland.