Climate changes and human activities combine to cause hydrological alterations, exerting tremendous influences on water conditions of the regional ecological environment. The increasing water use and deficiency of awareness of the ecological water have contributed to the shortage of ecological flow in the Yellow River basin. Thus, hydrological alterations should be taken into account in the estimation of the ecological instream flow. In this study, the t test and the Mann-Whitney U test are applied to analyze the ecological instream flow based on the monthly streamflow data extracted from the 7 hydrological stations along the main stem of the Yellow River basin. To assure the reliability of the change pointed detected in this study, the changing point is believed to be reliable only when the results of these two testing methods are in good agreement. Since a time series may have several change points, successive hypothesis test is used to identify all possible change points. In addition, possible causes behind the hydrological alterations are also discussed based on the publications. The changing points obtained are in good line with the time when the construction of the dams was done and also when other anthropogenic factors exert influences on the hydrological processes. After the detection of the change points, the ecological flow is defined as the streamflow with the maximum possibilities before the first change point. It is true that the ecological flows in various months are different in that the probability distributions of the streamflow components in different months are not the same. Therefore, the ecological flow should be considered at the monthly scale. The Pearson III, the general extreme value (GEV), the log normal (LOGN), and the Wakeby distributions are used to analyze the probability behaviors of the streamflow series and the parameters of the probability distributions are estimated with the L-moment technique. The probability distribution function with the highest goodness-of-fit after the Kolomogorov-Smirnov (KS) test will be accepted to evaluate the ecological flow. The results indicate that: (1) the ecological streamflow evaluated in this study based on the monthly instream flow data is reasonably right based on comparison with the results by the Tennant method, the minimum monthly average flow method, and the monthly frequency calculation method; (2) the streamflow greatly decreased after the change points due to climate change and anthropogenic activities; (3) the frequencies that the fluvial streamflow satisfies the ecological water requirements are decreasing; (4) the intensifying human activities such as human withdrawal of freshwater from the upper to the lower Yellow River basin are the major causes behind the decreasing water quantity; (5) the frequencies that the monthly streamflow satisfies the ecological water after the hydrological alteration should be kept at the same level as that before the hydrological alteration. Due to the climate changes and the booming development of the socio-economy, significantly enhancing water usage is observed, and it is particularly true in the lower Yellow River basin. In this sense, it is almost impossible for the fluvial streamflow to satisfy the water demand of the ecosystem for each month. On the other hand, the local ecosystem has acclimatized itself to the hydrological condition after the hydrological alterations. Thus, it could be feasible to keep the possibility that the fluvial streamflow may satisfy the water requirements of the ecological system at the same level as that before the hydrological variation. The results of this study could be of scientific and practical merits in terms of basin-scale water resources management.