The effects of operational parameters such as carbon source, C/N ratio, initial nitrate concentration, temperature, and pH value on heterotrophic denitrification and functional denitrifying genes were evaluated. When methanol was used as the sole carbon source, complete denitrification was performed in a short time without nitrous oxide (N2O) emission. Complete denitrification was performed at high C/N ratios (5.14 and 12.85) and low initial nitrate concentrations (75.9 and 151.6 mg N L−1). The denitrification rate was not temperature-sensitive in the range of 25–35 °C, but tended to decrease at a low pH of 5–6. The relationships between N2O emission and functional genes under various operational conditions were investigated by Pearson correlation and association network analyses. The C/N ratio was a key factor for N2O emission during the heterotrophic denitrification process. This information on the denitrification performance and its association with functional gene dynamics under various operational conditions is useful for N2O mitigation strategies for wastewater treatment processes.