Ammonia (NH3) volatilization is the main pathway, by which nitrogen (N) losses from calcareous and alkaline soils occur, and is responsible for economic losses and environmental hazards. We hypothesize that the application of biochar and urease inhibitor (NBPT) can mitigate NH3 production from urea by reducing its hydrolysis and the abundance of related microbial genes. To clarify this hypothesis, soil N fluxes and the genes controlling hydrolysis and nitrification were investigated under the combined application of urea (0, 75, 150, 225 and 300 kg N ha− 1 ), biochar (7.5 Mg ha− 1 ) and NBPT (2 %) for maize under field conditions. Soil moisture, temperature, pH, mineral N, NH3, urease activity (UA) and N cycle related genes (ureC, amoB and amoA) were quantified each 6–17 days during two growing seasons. The combined application of NBPT and biochar reduced NH3 by 13% of the control, which is twofold higher than the separate additions. All investigated parameters were raised with increasing N rate except pH, which was reduced. Biochar and NBPT mediated these effects of N fertilizer by increasing the soil pH, genes abundance, ammonium and nitrate contents, and reducing urea hydrolysis. Biochar maximized the ammonium and nitrate contents one week and three weeks following the N urea application, respectively. Accordingly, NH3 and UA peaked directly after N fertilization; nevertheless, NBPT delayed the peaks for three weeks and reduced their intensity by 24 % and 36 %, respectively, under the addition of 300 kg N ha− 1 . NBPT reduced ureC and amoB genes abundance by 14 % and 8 %, respectively, and increased amoA by 3 %. Biochar increased amoA and amoB abundance and nitrate content, while reduced the ureC gene. Biochar and/or NBPT reduced the negative effects of the soil pH on ureC gene. NBPT mitigated the reduction effects of pH on the amoB and amoA genes. In conclusion, NBPT mitigated NH3 emission by decreasing the ureC gene and UA, whereas biochar had a synergistic effect with NBPT by increasing amoB and amoA abundance and accelerating nitrification