Nitrate dosing is commonly used for controlling hydrogen sulfide in sewer systems. However, it may potentially facilitate N2O emission due to the denitrification process promoted by nitrate addition. In this study, lab-scale sewer reactors were operated to investigate the impact of nitrate addition on N2O production in sewer systems. Results showed that the N2O flux even increased by six times with the addition of nitrate when dissolved oxygen (DO) in the wastewater exceeded 0.4 mg/L. Principal component analysis showed that the N2O concentration was notably affected by DO and oxidation-reduction potential (ORP) in the wastewater. Furthermore, it was founded that N2O flux had a strong linear relationship with the DO concentration in the batch test. The microbial analysis found that the nosZ possessing organisms decreased significantly in the micro-aerobic condition and the copy numbers of nosZ gene declined consequently. It indicated that the inhibition of N2O reduced to N2 was responsible for significant accumulation and emission of N2O in the micro-aerobic condition. Given the gravity sewers are not completely anaerobic, the DO concentration is ranged from 0.1 to 2.4 mg/L in gravity sewers with the partially filled flow. Therefore, more attention should be paid to the N2O production when nitrate dosing for hydrogen sulfide controlling in gravity sewers.