Photoelectrotrophic denitrification (PEDeN) using bio-hybrids has the potential to remove nitrate (NO3-) from wastewater in an economical and sustainable way. As a gas of global concern, the mechanisms of nitrous oxide (N2O) emissions during this novel process remain unclear. Herein, a self-photosensitized bio-hybrid, i. e., Thiobacillus denitrificans-cadmium sulfide, was constructed and the factors affecting N2O emissions during PEDeN by the bio-hybrids were investigated. The system was sensitive to the input NO3--N and NO2--N, resulting in changes in the N2O/(N2+N2O) ratio from 1% to 95%. In addition to free nitrous acid (FNA), reactive oxidative species (ROS) were a unique factor affecting N2O emission during PEDeN. Importantly, the N2O reduction step exhibited greater susceptibility to the ROS than nitrate reduction step. The contributions of hydrogen peroxide (H2O2), superoxides (O2-•), hydroxyl radicals (•OH) and FNA to the inhibition of N2O reduction were >15.0%, >5.4%, 1.3%, and <70.2%, respectively for a reduction of 13.5 mg/L NO3--N. A significant down-regulation of the relative transcription of the gene nosZ demonstrated that the inhibition of N2O reductase occurred at the gene level. This finding has important implications not only for mitigating N2O emissions during the PEDeN process but also for encouraging a reexamination process of N2O emissions in nature, particularly in systems in which ROS are present during the denitrification process.,Copyright © 2020 Elsevier Ltd. All rights reserved.