【Objective】Nitrous oxide (N2O) is a potent greenhouse gas with a global warming potential 296 times that of carbon dioxide (CO2). Microbial-driven nitrification and denitrification are major processes contributing to N2O production. While numerous studies have explored the combined effects of biochar and organic fertilizer, most have been short-term, and the legacy effects of aged biochar on soil N2O emissions remain poorly understood. The interactive effects of its combined application with organic fertilizers necessitate further investigation. 【Method】Soil samples were obtained from a seven-year field experiment comprising four distinct treatments: (1) control (urea application, F); (2) one-time basal application of biochar (FB); (3) annual application of organic fertilizer (OF); and (4) combined annual application of organic fertilizer and one-time basal biochar (OFB). In the organic fertilizer treatments, 25% of the urea nitrogen was substituted with organic fertilizer nitrogen. A laboratory incubation experiment was conducted to measure cumulative N2O emissions, quantify the abundances of key functional genes (including nirS, nirK, and nosZ), and partition the relative contributions of fungal and bacterial pathways to N2O emissions. 【Result】The result showed that compared to the control, cumulative N2O emissions were significantly reduced by 49.4% in the biochar treatment (FB), 38.4% in the organic fertilizer treatment (OF), and 59.3% in the combined treatment (OFB). Biochar significantly decreased the fungal contribution to N2O emissions (FDC) by 11.4% and increased the bacterial contribution (BDC) by 5.8%. Organic fertilizer reduced the contribution of ammonia-oxidizing bacteria (AOB) by 15.3% but increased the bacterial contribution by 12.1%. The combined application of biochar and organic fertilizer decreased the fungal contribution by 9.7% and increased the bacterial contribution by 15.7%. Structural equation modeling (SEM) indicated that biochar directly reduced FDC and enhanced BDC, organic fertilizers significantly enhance BDC and reduce (nirS+nirK)/nosZ, thereby decreasing N2O emissions. 【Conclusion】These results demonstrate the sustained potential of biochar and organic fertilizer amendments in reducing greenhouse gas emissions from agricultural soils and provide mechanistic insights into how these amendments regulate microbial processes governing N2O production. This research outcome provides scientific support for in-depth analysis of the legacy effects of biochar and organic fertilizer application on soil and their microbiological mechanisms offering scientific guidance for optimizing fertilization practices to achieve the goal of reducing N2O emissions from farmland soils.