Abstract: Triphenyl phosphate (TPhP), a typical organophosphate ester (OPE), has garnered significant attention due to its widespread occurrence in surface water environments and associated high ecological risks. Investigating its effects on rhizosphere ammonia-oxidizing microorganisms in aquatic ecosystems is crucial for understanding the response of nitrogen-cycling microbes to organic pollutant stress and thereby providing theoretical support for N₂O mitigation strategies. In this study, Phragmites australis was used as the model plant to investigate the impacts of TPhP at environmentally relevant concentrations on rhizosphere ammonia oxidation and N₂O emission. Real-time quantitative PCR (qPCR) and metagenomic sequencing were employed. The results show that under TPhP stress, the abundance of complete ammonia-oxidizing bacteria (comammox, CMX) significantly increased, while the metabolic activities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were markedly suppressed. TPhP promoted the conversion of ammonium to nitrate in the rhizosphere and substantially reduced the N₂O emission flux from the plant cultivation system. From the perspective of ammonia oxidation rates, the contribution of AOA was almost negligible. In contrast, the ammonia oxidation rates of CMX and AOB were consistent with their abundance changes: the CMX ammonia oxidation rate increased significantly, whereas that of AOB decreased markedly. This trend effectively suppressed the potential for N₂O production. Furthermore, metagenomic analysis revealed that TPhP enhanced the synergistic interactions among rhizosphere microorganisms and upregulated the expression of the ammonia monooxygenase (amo) gene, promoting the growth of the CMX genus Nitrospira, while inhibiting the AOA genus Nitrosarchaeum and the AOB genus Nitrosomonas. This study can not only provide data to comprehensively understand the impacts of TPhP on nitrogen-cycling microorganisms, but also offer new insights into the functions of CMX under TPhP stress.