Biochar soil amendments are attracting the increased attention as one strategy to improve soil microbially ecological environment and regulate soil nitrogen cycle. The rainfed maize cropland in the western Henan was established to study the effects of biochar application rates (T0:0 t/hm², T1:20 t/hm², and T2:40 t/hm²) on soil physicochemical properties and N₂O emission fluxes using the static chamber/gas chromatography method, and the function marker genes of arbuscular mycorrhizal (AM) fungi, ammonia monooxygenase gene (amoA), nirK, nirS and nosZ, which were responsible for nitrification and denitrification using real-time fluorescence quota PCR. The results showed that the soil pH and water content increased, soil organic carbon, total nitrogen and ammonium nitrogen content increased significantly, and soil bulk density and nitrate nitrogen content decreased significantly with increasing biochar applied. Compared with T0 treatment, soil organic carbon increased significantly by 38.44% and 71.01% under T1 and T2 treatments, respectively; soil ammonium nitrogen content increased significantly by 15.89% and 30.46% under T1 and T2 treatments, respectively; soil total nitrogen content increased significantly by 14.87% under T2 treatment; nitrate nitrogen content decreased by 10.57% and 21.40% under T1 and T2 treatments, respectively. Moreover, the AM fungi colonization rate and nosZ gene abundance increased significantly, and AOA, AOB, nirK and nirS gene abundance decreased significantly with increasing biochar applied. Compared with T0 treatment, AM fungi increased significantly by 71.88% and 115.88% under T1 and T2 treatments, respectively. The N₂O emission fluxes and emission accumulations under biochar addition were all lower than those under no biochar, ranked as T0 > T1 > T2. The correlation analysis showed that the abundance of AM fungi was significantly positively (P<0.05) correlated with the biochar rate, and the abundance of nosZ gene and biochar rate was significant. There was a significantly negative correlation between AOA, AOB, nirK, nirS gene abundance and biochar rate (P<0.01). The N₂O emission fluxes showed a significantly positive correlation (P<0.01) with AOA, nirK, nirS, soil water content and nitrate nitrogen, but significantly negatively (P<0.01) correlated with AM fungi, nosZ, ammonium nitrogen and extractable glomalin related soil protein content. Aggregated boosted trees (ABT) analysis showed that the relative influence of AOA on N₂O emission fluxes was the largest, followed by AM fungi and nirK. In summary, the application of biochar improved soil physicochemical properties, increased soil AM fungi colonization rates, regulated nitrification and denitrification functional genes, and reduced N₂O emission, which provided a theoretical basis for the rational application of biochar and reduction of N₂O emissions in rainfed maize cropland.