Emission of nitrous oxide (N2O), a greenhouse gas, is of growing concern in biological wastewater treatment. N2O emission from biofilm in a one-stage completely autotrophic nitrogen removal system was investigated using microelectrodes in this study. It is indicated that the pathways of nitrogen transformation in biofilm mainly included partial nitrification and anaerobic ammonium oxidation (anammox), also included nitrification and heterotrophic denitrification (HD). Ammonium-oxidizing bacteria (AOB) denitrification and HD were the main pathways resulting in N2O production in the biofilm, and hydroxylamine (NH2OH) oxidation was a subordinate pathway. In addition, the amount of N2O emission in test in which both NH4+ and NO2- were added (NH4+-N: NO2--N = 1:1) was about 2 times greater than that in test with NH4+ addition only. This result expressed that NO2- is an important factor affecting N2O production in the biofilm. In conclusion, the present study provides a theoretical support for reducing N2O production in one-stage completely autotrophic nitrogen removal system. (C) 2017 Elsevier Ltd. All rights reserved.