Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern. This paper reports findings of the effects of carbon/nitrogen (C/N) ratio on N2O production rates in a laboratory-scale biological aerated filter (BAF) reactor, focusing on the biofilm during nitrification. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and microelectrode technology were utilized to evaluate the mechanisms associated with N2O production during wastewater treatment using BAF. Results indicated that the ability of N2O emission in biofilm at C/N ratio of 2 was much stronger than at C/N ratios of 5 and 8. PCR-DGGE analysis showed that the microbial community structures differed completely after the acclimatization at tested C/N ratios (i.e., 2, 5, and 8). Measurements of critical parameters including dissolved oxygen, oxidation reduction potential, NH4+-N, NO3--N, and NO2--N also demonstrated that the internal micro-environment of the biofilm benefit N2O production. DNA analysis showed that Proteobacteria comprised the majority of the bacteria, which might mainly result in N2O emission. Based on these results, C/N ratio is one of the parameters that play an important role in the N2O emission from the BAF reactors during nitrification.