It is important to manage water resources of the upper Yellow River basin for the new Silk Road economic belt. In recent decades, under the combined human activities and influence of climate, the hydrologic regime of the upper Yellow River basin shows remarkable variations that have caused many issues. So potential human indicated influence has been drawing increasing attention from hydrologists and local governments. The aim of this study is to determine the changes in the hydrological characteristic parameters and mean annual runoff series of the upper Yellow River basin. This paper took the representative Lanzhou Station in the upstream Yellow River as an example, used the TFPW-MK mutation test and rank sum test to analyze the location of the variation points of hydrological series. By contrasting two different analysis results between natural and measured river runoff series, the impacts of human activities on the long-term hydrological regime were investigated. The variation range of hydrological ecological indexes before and after variation were analyzed by the method of indicators of hydrologic alteration (IHA). The results show: a) the hydrological series of Lanzhou Station is a significant decreasing trend can be observed in the natural stream flow series in 1985 with a high degree of hydrological variability, while human activities play an important role; b) various ecological indicators have changed in different degrees that have caused the deterioration of ecological conditions around 1985; and c) continuous decreasing stream flow in the upper Yellow River basin will trigger serious shortages of fresh water in the future, which may challenge the sustainability and safety of water resource development in the river basin, and should be paid great attention before 2020. Variation analysis and diagnosis of eco-hydrological indexes in the upper reaches of the Yellow River can provide a basis for the development, utilization, and protection of water resources in this area.