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In subtropical regions, we have an incomplete understanding of how long-term tillage, stubble, and nitrogen (N) fertilizer management affects soil biological functioning. We examined a subtropical site managed for 50 years using varying tillage (conventional till (CT) and no-till (NT)), stubble management (stubble burning (SB) and stubble retention (SR)), and N fertilization (0 (N0), 30 (N30), and 90 (N90) kg ha⁻¹ y⁻¹) to assess their impact on soil microbial respiration, easily extractable glomalin-related soil protein (EEGRSP), and N mineralization. A significant three-way tillage × stubble × N fertilizer interaction was observed for soil respiration, with NT+SB+N0 treatments generally releasing the highest amounts of CO₂ over the incubation period (1135 mg/kg), and NT+SR+N0 treatments releasing the lowest (528 mg/kg). In contrast, a significant stubble × N interaction was observed for both EEGRSP and N mineralization, with the highest concentrations of both EEGRSP (2.66 ± 0.86 g kg⁻¹) and N mineralization (30.7 mg/kg) observed in SR+N90 treatments. Furthermore, N mineralization was also positively correlated with EEGRSP (R² = 0.76, <i>p</i> < 0.001), indicating that EEGRSP can potentially be used as an index of soil N availability. Overall, this study has shown that SR and N fertilization have a positive impact on soil biological functioning.