Estuaries as the coastal habitats connecting rivers to the sea are important carbon and nutrient sinks of the surrounding systems and highly productive ecosystems with multiple ecosystem services. However, most estuarine habitats have been intensively modified and highly degraded due to urbanisation for the purposes of enhancing flood control and increasing human land use. Engineering work such as channelisation and straightening of the estuary-associated rivers commonly result in salinity intrusion and increased tidal influence to biotic community. Eutrophication caused by various anthropogenic activities could lead to changes in assemblages of primary producers in urbanised estuaries and could alter the food web structure. Due to the limited studies on the influence of such salinity intrusion and eutrophication on the biological community structure, little is understood for the shift of trophic basis of urbanised estuary ecosystems in response to human impacts. The strong seasonality of subtropical precipitation pattern could also complicate the effects of salinity and nutrient enrichment on the estuarine food web structure. In this study, three study sites were selected along the tidal main reach of the urbanised Danshuei River estuary in the highly developed northern Taiwan. Stable carbon and nitrogen isotope analysis and gut content analysis were used to determine the trophic basis of the omnivorous fishes which represented the most dominant group of consumers and could be directly/indirectly affected by the change of primary producer assemblage in response to effects of salinity, nutrient enrichment and seasonality. Thus, the stable isotope mixing model (SIAR) was employed to evaluate the relative contribution of various basal energy sources assimilated by the omnivorous fish consumers. Our results will be further discussed to address the effects of anthropogenic impacts on food web structure in an urbanised estuary.