Yushugou River basin of East Tianshan Mountains receives water from melting glaciers. In recent years, the glaciers retreated strongly due to global warming which intensified the water cycle in the river basin. For this reason, the relation of water bodies based on hydrochemistry and isotope in the summer flood was carried out. Hydrochemistry research showed that there was frequent hydraulic interaction between river water and groundwater. Studying the isotopes and Cl− of river water, glacier meltwater, groundwater and precipitation, indicated that Yushugou River was recharged by the glacier meltwater, groundwater and precipitation during the summer flood period. The analysis result based on the three-component mixing model showed that Yushugou River was recharged by 54.9% of glacier meltwater, 37.6% of the run-off came from groundwater, while less than 8% was contributed by precipitation. The study suggests that the role of glacier meltwater and groundwater, especially glacier meltwater, should be specially concerned in water resource protection and reasonable utilization, and the injection of glacier meltwater is the main reason for run-off variation in this alpine basin during the summer flood period.