The subalpine shrub zone of the Qilian Mountains is an important water-retaining area, and it is crucial to clarify the processes of its hydrological cycle. Therefore, based on the stable isotope values of different water bodies in the subalpine shrub zone of the eastern Qilian Mountains from May to October 2019, the characteristics of δD and δ18O of different water bodies and their hydraulic relationships with each other were studied. The results showed that the stable isotope values of precipitation exhibited the largest fluctuations, while they were the most stable for groundwater. Plant transpiration was stronger than the evaporation of other water bodies. The stable isotope of precipitation was enriched in high temperature and low humidity environments. Isotopic values of plant and soil water were higher and more stable on the semi-sunny slope than on the semi-shady slope. According to the stable isotopes, there was a strong hydraulic relationship between the different water bodies in the study area, and precipitation was the ultimate source of all of them. Precipitation replenished soil water through infiltration. Part of the soil water was absorbed by plants, while the rest continued to infiltrate to replenish groundwater. Groundwater and precipitation replenished the river water.