In this study, the heterotrophic nitrification–aerobic denitrification strain JQ1004 was investigated in terms of its nitrogen removal mechanism and kinetic properties, laying the foundation for its application in the field of wastewater treatment. Nitrogen balance analysis revealed that the final metabolic product was N₂, and approximately 54.61% of N was converted into cellular structure through assimilation. According to the fitting of the Compertz model, the maximum degradation rates of ammonia and nitrate were 7.93 mg/(L·h) and 4.08 mg/(L·h), respectively. A weakly alkaline environment was conducive to N removal, and the sensitivity of functional genes to acidic environments was amoA > nirS > narG. An appropriate increase in dissolved oxygen significantly enhanced heterotrophic nitrification activity, and notably, the denitrification-related functional gene narG exhibited greater tolerance to dissolved oxygen compared to nirS. The transcription level of amoA was significantly higher than that of narG or nirS, confirming that there might have been direct ammonia oxidation metabolic pathways (NH₄⁺→NH₂OH→N₂) besides the complete nitrification and denitrification pathway. The annotation of nitrogen assimilation-related functional genes (including gltB, gltD, glnA, nasA, nirB, narK, nrtP, cynT, and gdhA genes) in the whole-genome sequencing analysis further confirmed the high assimilation nitrogen activity of the HN-AD strain.