Growing human activities in recent decades have collectively resulted in large amounts of nutrients export into coastal oceans. As the most reactive nitrogen species, ammonium ( NH4+) plays the critical role in biogeochemical cycles in estuaries and the coastal ocean. In the highly polluted Pearl River Estuary (PRE), NH4+ predominates to be the energy source for nitrification and to be the material source for bacteria and phytoplankton to grow. Both above processes are affected by light, yet in opposite ways. Nevertheless, rare studies paid attention to dual NH4+0.25em transformation processes specifically during dark conditions. By using nitrogen isotope tracer technique, we quantitatively and simultaneously differentiated two distinctive NH4+ consumption pathways, that is, NH4+ oxidation (AOD) and assimilation (AAD) rates, especially under dark conditions along the PRE during the 2015 and 2017 summer cruises when biological activities were the highest. We found the NH4+ transformations display a bilayer structure with AAD > AOD in almost all the surface waters and vice versa in all bottom waters, suggesting bacteria and phytoplankton (mainly bacteria) control NH4+ consumption in surface during the night while nitrifiers are the major NH4+ consumer in the bottom waters. Through redundancy analysis, we found that both processes are mainly driven by NH4+ in the PRE during summer. In addition, in the downstream PRE during two cruises, AOD at most contributed 27% and 8% of the water column community dissolved oxygen (DO) consumption assuming the Redfield stoichiometry has no direct effect on hypoxia formation in the PRE during summer.