The accelerated melting of glaciers in the Tianshan Mountains of Central Asia, driven by global warming and heightened regional precipitation, has significantly altered mountain runoff dynamics and attracted widespread attention. The Aksu River is the largest river on the southern slopes of the Tianshan Mountains, nourishing the largest oasis in the arid region of southern Xinjiang, northwest China. This study analyzed 844 water samples from the Aksu River Basin, including glacier meltwater, river water, groundwater, and precipitation, to investigate stable isotopic characteristics and runoff composition, thereby enhancing the understanding of the basin's hydrological processes. By calculating the Local Meteoric Water Line, Local Evaporation Line, D-excess, and lcexcess, this study confirmed the intense evaporation affecting runoff and groundwater in the basin and the subcloud evaporation of precipitation from an isotopic perspective. The runoff composition was quantified using Bayesian Monte Carlo (BMC) isotope mixing model, revealing that glacier meltwater is the primary contributor to the river during the ablation period, while precipitation plays a critical role in runoff during the non-ablation period. Furthermore, by comparing end-member mixing analysis (EMMA) with the BMC model, this study demonstrated the accuracy of the BMC approach and highlighted its advantages. A sensitivity analysis was also performed to assess the effect of input uncertainties on the model outputs.