Based on the daily precipitation data during 1960–2016 at 72 stations and the daily streamflow data during 1956–2016 at 7 hydrological stations in the Huai River Basin (HRB), China, eco-surplus and eco-deficit under influences of abrupt streamflow behaviors were analyzed using Flow Duration Curve (FDC). The relations between indicators of hydrological alteration (IHA) and ecological indicators (Shannon Index, SI) were quantified, investigating impacts of altered hydrological processes on the evaluations of the ecological instream flow. Besides, we also quantified fractional contributions of climatic indices to nonstationary ecological instream flow using the Generalized Additive Models for Location Scale and Shape (GAMLSS) framework. While the possible impact of human activities on ecological instream flow will be revealed based on land use changes data. The results indicated that: (1) FDC is subject to general decrease due to hydrological alterations, and most streamflow components are lower than 25% FDC. We found increased eco-deficit and decreased eco-surplus due to altered hydrological processes. The FDC of the streamflow in the main stream of the HRB is lower than that along the tributaries of the HRB. Eco-surplus (eco-deficit) changes are in good line with precipitation anomaly changes during the Spring, Autumn and Winter periods. However, the hydrological alterations due to hydrological regulations by the reservoirs are the primary cause behind the mismatch between ecological instream flow and precipitation anomalies during summer; (2) Annual and seasonal eco-surplus (eco-deficit) is decreasing (increasing) and that during winter season is an exception. Although higher eco-surplus in winter than in other seasons, the eco-surplus is decreasing persistently and the 21st century witnessed the lowest eco-surplus along the main stream of the HRB. Meanwhile, the Shannon index indicated decreased ecological diversity across the HRB; (3) The ecological instream flow is highly sensitive to The Pacific Decadal Oscillation (PDO), North Atlantic Oscillation (NAO) and Niño 3.4 Sea Surface Temperature Index (Nino3.4). Meanwhile, the ecological instream flow along the mainstream of the HRB is highly sensitive to climate indices. While the ecological instream flow by GAMLSS model has better fitting performance in describing the extreme values and local trends.