Mountain glaciers are considered natural indicators of warming and a device for climatic change. In addition, it is also a solid reservoir of freshwater resources. Along with climate change, clarifying the dynamic changes of glacier in the Aksu River Basin (ARB) are important for hydrological processes. The study examined the variations in glacier area, elevation, and their reaction to climate change in the ARB between 1990 and 2022. The glacier melt on the runoff is explored from 2003 to 2020. This investigation utilized Landsat and Sentinal-2 images, ICESat, CryoSat, meteorological and hydrological data. The findings suggest that: (1) The glacier area in the ARB retreated by 309.40 km2 (9.37%, 0.29%·a−1) from 1990 to 2022. From 2003 to 2021, the ARB glacier surface elevation retreat rate of 0.38 ± 0.12 m·a−1 (0.32 ± 0.10 m w.e.a−1). Comparison with 2003–2009, the retreat rate is faster from 2010 to 2021. (2) From 1990 to 2022, the Toxkan and the Kumalak River Basin’s glacier area decreases between 61.28 km2 (0.28%·a−1) and 248.13 km2 (0.30%·a−1). Additionally, the rate of glacier surface elevation declined by −0.34 ± 0.11 m·a−1, −0.42 ± 0.14 m·a−1 from 2003 to 2021. (3) The mass balance sensitivities to cold season precipitation and ablation-phase accumulated temperatures are +0.27 ± 0.08 m w.e.a−1(10%)−1 and −0.33 ± 0.10 m w.e.a−1 °C−1, respectively. The mass loss is (962.55 ± 0.57) × 106 m3 w.e.a−1, (1087.50 ± 0.68) × 106 m3 w.e.a−1 during 2003–2009, 2010–2021 respectively. Warmer ablation-phase accumulated temperatures dominate glacier retreat in the ARB. (4) Glacier meltwater accounted for 34.57% and 41.56% of the Aksu River’s runoff during the ablation-phase of 2003–2009 and 2010–2020, respectively. The research has important implications for maintaining the stability of water resource systems based on glacier meltwater.