Identifying microbial yield and the pathway relating to nitrous oxide (N₂O) production in nitrification were essential prerequisites for modeling N₂O production and emissions. A thermodynamic method based on standard Gibbs free energy change of bio-chemical reaction was used to evaluate the NH₃-NO, NH₃-NO₂⁻ , NH₄⁺-NO, NH₄⁺-NO₂⁻ , NH₂OH-NO, and NH₂OH-NO₂⁻ reactions. Meanwhile, the stoichiometric equations of the relevant reactions were built up to ascertain the microbial yield of the microorganisms responsible for N₂O production. The results indicated that: 1) all the above reactions had the possibility of producing N₂O in nitrification as the standard Gibbs free energy changes of all the reactions were negative, 2) all the possible reactions can be used as the pathways to describe N₂O production in nitrification in mathematical models, and 3) theoretically, the yield coefficient of autotrophs relating to N₂O production was in the range of 0.097~0.194 g cell/ g N. Ammonia-oxidizing bacteria were responsible for the production of N₂O in nitrification. In order to develop a unified model for better simulation of N₂O production and emissions in the biological wastewater treatment process, intensive studies should be carried out to reveal undiscovered pathways relating to N₂O production.