Biochar application to soil is currently widely advocated for a variety of reasons related to sustainability. However, the synergistic effects of biochar combined with mineral or organic fertilizer on soil N2O emissions, NH3 volatilization, and plant N uptake are poorly documented. Field plot experiments planted with peanut were conducted under the application of biochar (derived from rice husk and cottonseed husk, 50 t ha-1) with organic or mineral fertilizer. It was found that biochar increased soil nutrient availability and decreased surface soil bulk density, demonstrating that biochar could improve the soil quality especially in the 0-20-cm profile. The total N content of the plant changed little with treatments, but the kernel N concentration increased significantly when biochar was applied with organic fertilizer. Peanut yield increased with biochar amendment while no significant difference was observed in plant biomass, suggesting biochar had a positive effect on belowground biomass. Peanut N uptake was also increased following biochar amendment with either organic or mineral fertilizers. While biochar amendment had no significant effect on soil NH3 volatilization, it did decrease the cumulative N2O emission by 36.3% on average with organic fertilizer, and by 32.6% with mineral fertilizer, respectively (p < 0.05). The copy numbers of 16S rDNA, nifH, nirK, and nirS were not influenced by the application of biochar; however, the copy number of nosZ was significantly increased under biochar plus mineral fertilizer treatment. The results imply that biochar application can suppress N2O emissions, as a result of abiotic factors and enhanced peanut N uptake rather than changes of denitrification genes.