Due to the lack of regular monitoring networks and practical assessment criteria for contaminants of emerging concern in rivers, it is challenging to quantify the influences of human activities on their occurrence and ecological impacts on the estuarine environment. To address this issue, herein, organophosphate esters (OPEs) were selected and tested in the Pearl River Estuary (PRE), which is an urban estuary surrounded by a megalopolis (the Greater Bay Area) in South China. A field-based investigation was conducted on the riverine outlets of the PRE by considering tidal and seasonal variations coupled with models to improve the assessment of human-derived stress on this urban estuary. The results indicated that the occurrence of OPEs was strongly influenced by emissions from land-based sources and hydrological conditions with minor influences from biogeochemical processes. Approximately 45,200 kg year −1 of ∑ 12 OPE was discharged from the eight major riverine outlets to the PRE in 2020−2021. Tributyl phosphate and tris(2-chloroethyl) phosphate with the highest environmental stress index are the two priority OPEs, necessitating further monitoring and control measures in this estuary. The present study demonstrates that the urbanized riverine outlets face greater environmental stress and lower resilience to OPE pollution than other less-urbanized outlets.