Maintaining natural hydrologic variability is essential in conserving native riverine biota and river ecosystem integrity. Hydrologic regimes play a major role in structuring the biotic diversity within river ecosystems, as they control key habitat conditions within the river channel, the floodplain, etc. Alterations in streamflow regimes may modify many of these habitat attributes and impair ecosystem connectivity. There are many dams constructed in the Huaihe River basin that are drastically altering the natural hydrologic regimes of the river. We selected the Bengbu Sluice as a control node to study the influence of the Bengbu Sluice and all its upstream dams on the hydrologic regime. Using Indicators of Hydrologic Alteration and Range of Variability Approach methods, we assessed hydrologic alteration at the streamgauge site to demonstrate the influence of dams on ecohydrological conditions in the Huaihe River basin. The results show that dams have a strong influence on ecohydrological conditions, especially in dry seasons. The river ecohydrological targets and the minimum ecological and environmental flow requirements for the Bengbu section defined by this study can support ecosystem management and restoration plans and provide ecological operations for the Bengbu Sluice.