Abstract: Dam-induced hydrological alterations and related ecological problems have been arousing considerable concern from hydrologists, ecologists, and policy-makers. The East River basin in China is the major provider of water resources for mega-cities within the Pearl River Delta and meets 80% of annual water demand of Hong Kong. In this study, ecodeficit and ecosurplus were analyzed to determine the ecological impact of water impoundments. Also, Do and DHRAM were employed to evaluate the degree of alteration of hydrological regimes, and ERHIs were analyzed to evaluate the influence of hydrological alterations on ecological diversity. Results indicate that: (1) the magnitude and frequency of high flows decrease and those of low flows increase due to the regulation of reservoirs; (2) variations of annual ecosurplus are mainly the result of precipitation changes and the annual ecodeficit is significantly influenced by reservoirs. However, ecodeficit and ecosurplus in other seasons, particularly autumn and winter, are more influenced by reservoir regulation; (3) impacts of reservoirs on hydrological regimes and eco-flow regimes are different from one station to another due to different degrees of influence of reservoirs on hydrological processes at different stations. The longer the distance between a reservoir and a hydrological station is, the weaker the influence the water reservoir has on the hydrological processes; (4) ecodeficit and ecosurplus can be accepted in the evaluation of alterations of hydrological processes at annual and seasonal time scales. Results of Shannon Index indicate decreasing biological diversity after the construction of water reservoirs, implying negative impacts of water reservoirs on biological diversity of a river basin and this should arouse considerable human concerns. This study provides a theoretical background for water resources management with consideration of eco-flow variations due to reservoir regulation in other highly-regulated river basins of the globe.