The dynamic characteristics of N2O emissions and nitrogen transformation in a sequencing batch biofilm reactor (SBBR) using the completely autotrophic nitrogen removal over nitrite (CANON) process coupled with denitrification were investigated via 15N isotope tracing and thermodynamic analysis. The results indicate that the Gibbs free energy (ΔG) values of N2O production by the nitrifier denitrification and heterotrophic denitrification reactions were greater than that of NH2OH oxidation, indicating that N2O was easier to produce via either nitrifier and heterotrophic denitrification than via NH2OH oxidation. Ammonia-oxidizing bacteria (AOB) denitrification exhibited a higher fs0 (the fraction of electron-donor electrons utilized for cell synthesis) than NH2OH oxidation. Therefore, AOB preferred the denitrification pathway because of its growth advantage when N2O was produced by the AOB. The N2O emissions by hydroxylamine oxidation, AOB denitrification and heterotrophic denitrification in the SBBRs using different C/N ratios account for 5.4–7.6%, 45.2–60.8% and 33.8–47.2% of the N2O produced, respectively. The total N2O emission with C/N ratios of 0, 0.67 and 1 was 228.04, 205.57 and 190.4 μg N2O-N·g−1VSS, respectively. The certain carbon sources aid in the reduction of N2O emissions in the process.