Abstract: Biochar is a promising material for mitigating greenhouse gas emissions. In addition to carbon sequestration, it has positive effect on the ozone-depleting gas nitrous oxide (N₂O), which is with long residence time and strong warming potential. In this research effort, an anaerobic incubation experiment was conducted. Three treatments with different biochar application rates were set, taking account of biochar to soil ratio (w/w):0 (0BC), 1% (1%BC) and 5% (5%BC). Soil gravimetric water content was controlled at 20%. According to the robotized incubation platform providing real-time determination of N₂O and N₂ concentrations and soil denitrification functional gene abundance measurement, we analyzed the impact of biochar on N₂O consumption and biological mechanisms. The main results indicated that after a 20-hour anaerobic incubation, the denitrification functional gene abundance of 0BC treatment was 6.80×10⁷ (nirK), 5.59×10⁸ (nirS), 1.22×10⁸ (nosZ) gene copies per gram soil, respectively. Compared with 0BC treatment, the nirS gene abundance of 1%BC treatment increased from the initial 2.65×10⁸ to 7.43×10⁸ gene copies per gram soil, while, the nosZ gene abundance increased by an order of magnitude from 4.82×10⁷to 1.50×10⁸ gene copies per gram soil. However, there was no significant change in nirK gene abundance. And the denitrification functional gene abundance of 5%BC treatment did not show marked variations. In conclusion, the N₂/(N₂O+N₂) ratio of treatments with biochar application was clearly higher than 0BC treatment. The results of correlation analysis showed that nirS and nosZ gene abundance was significantly correlated with the N₂O concentration at 0.01 level, and the abundance of nirS and nosZ genes all increased as N₂O concentration declined at the end of the experiment. Therefore, in the present trial, a 1% biochar addition significantly increased the abundance of denitrifying bacteria with nirS and nosZ genotypes and N₂/(N₂O+N₂) ratio, and promoted the complete reduction of N₂O to N₂. The main mechanism of the biochar effect on N₂O emission was the enhanced reduction activities and gene expression of nosZ-containing microorganisms, resulting in complete denitrification.