The upper Yellow River Basin is the most important water source, which is of decisive significance to the water resources security, ecological environment and food security. Under the background of warm and humid climate in Northwest China, and climate, hydrology and ecology research have attracted in the upper Yellow River Basin. The multi-scale variation characteristics of climate and its impact on ecological vegetation and runoff are analyzed by using satellite remote sensing data, grid fusion data and hydrological monitoring data. The results was summarized as follows: (1) it is consistent with whole region on warming and wetting trend recent 40 years, meanwhile, which show significant regional differences, the temperature and precipitation increase rate was 0.023℃/a, 1.09mm/a respectively; in the past 40 years, the precipitation showed obvious increasing trend over the confluence area of Huangshui River Basin and central part of Gansu province, while the increasing trend in the desert region of flow area was significantly. The whole Yellow River Basin has experienced a significant wetting process since 2000. Moreover, the actual evapotranspiration increased significantly over confluence and flow area since 2000, but the source area decreased. (2) Due to the current warm and humid climate, which was conducive to the vegetation growth, since 1999, the vegetation increased significantly with rate reaching 0.04/10a in the source and confluence area. From the long-term trend, we concluded the previous year precipitation have significant influence on vegetation index in source and confluence area, however, the flow area vegetation index present positive correlation with previous precipitation. It is obvious that the precipitation improved the growth of vegetation, while the temperature have more complex effect on vegetation. thus, the different vegetation type may be the key factor causing the differences response to precipitation. (3) Affected by the variation of climate and vegetation, the annual runoff of both Tangnaihai and Lanzhou Station have been decreasing in the past 40 years, but showed significant increasing trend since 1998, and the rate of increased runoff of Lanzhou station is nearly three times that of Tangnaihai station. We found there was significant positive correlation between the runoff of Tangnaihai station and precipitation, while the correlation coefficient of Lanzhou Station was lower than that of Tangnaihai from long long-term trend. After detrend, precipitation is the most important factor to determine the annual runoff, meanwhile, we should not ignore the influences of ecological vegetation, frozen soil degradation, water reserve change and social activities on runoff. This study provides reference for the scientific response to the ecological protection and the realization of high quality development in the upper Yellow River Basin.