The variation extent of the eco-hydrological regime of the Weihe River Basin was assessed using the most ecologically relevant hydrologic indicators (ERHIs). Seven indicators were selected as the ERHIs of the Weihe River Basin using the principal component analysis method. The observation-simulation comparative analysis method based on the variable infiltration capacity model was used to quantitatively attribute the changes of the eco-hydrological regime of the Weihe River Basin. The results show that the runoff series at the Xianyang and Huaxian stations had a significant downward trend, and the change points of both runoff series appeared in 1990. The comprehensive alternation degrees of the ERHIs obtained from the measured runoff series were 41% and 34%, respectively, at the Xianyang and Huaxian stations, and those obtained from the simulated runoff series at the two stations were both 18%. Climate change and human activities caused the variations of eco-hydrological regime in the basin, and their contribution rates to the overall hydrological alternation degree of ERHIs were 44% and 56% at the Xianyang Station and 53% and 47% at the Huaxian Station.