As an important hydrological ecosystem component, the glacier basin has great significance for climate and environment, and it is also linked to regional water sustainability. In this paper, the sampling and isotope analysis of glacial ice, ice-melt water, river water (river midstream and river downstream), groundwater (spring), and precipitation were carried out in a hydrological year of the Mingyong Glacier basin, which is located at the Meili Snow Mountains, Southeastern Tibetan Plateau. At the same time, the hydrograph separation of the recharge sources of the lower mountain pass is studied. The results show that the range of δD, δ18O, and d-excess (deuterium excess) in natural water bodies are significantly different, and the precipitation is the most obvious. The high values of δD and δ18O in the water samples all appeared in spring and summer, and the low values appeared in autumn and winter, while glacial ice showed opposite trends. Meanwhile, the local meteoric water line (LMWL) of the Mingyong Glacier basin is δD = 8.04δ18O + 13.06. The End-Member Mixing Analysis (EMMA) was adopted to determine the sources proportion of river water (river downstream) according to the δD, δ18O, and d-excess ratio relationships. The results showed that the proportion of ice-melt water, groundwater, and precipitation in the ablation period was 80.6%, 17.2%, and 2.2% as well as 19.2%, 73.1%, and 7.7% in the accumulation period, respectively. Ice-melt water has a higher conversion recharge rate to groundwater and indirectly recharges river water, especially in nonmonsoon seasons. In other words, the main recharge source of river water in the lower reaches of the Mingyong Glacier basin during the ablation period is ice-melt water. In the accumulation period, the main recharge source of river water in the lower reaches of the Mingyong Glacier basin is groundwater, while nearly half of the recharge of groundwater comes from ice-melt water. Therefore, regardless of the ablation period or the accumulation period, ice-melt water is sustainable and important to this region.