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Aquatic ecosystems require ecological water allocation to prevent from being damaged by natural disasters and undue exploitation. This paper discusses and estimates the ecological water requirements (EWRs) of typical areas in the Huaihe Basin to determine rational allocations of water resources and promote regional improvements of the ecological environment. The main river course, including Hongze Lake and Nansi Lake, was selected as the study subject. Calculational methods for the river and lake EWRs were based on the reasonableness of the results and data availability. The monthly guarantee rate method was used to calculate monthly, flood period, non-flood period, and annual EWRs for the main river course and the main tributaries at two different guarantee rates. The minimum water level method was used to calculate annual EWRs for Hongze Lake and the upper and lower Nansi Lake of 1.521 × 109 m3, 0.637 × 109 m3, and 0.306 × 109 m3. The results were used to evaluate the rationality of the quantity of water resources allocated to ecological uses in the Huaihe Basin during 1998–2003. The result shows that the present water resource allocations in the Huaihe Basin cannot satisfy the basic ecological requirements for some years, especially years with less precipitation.