Abstract: In Northwest China, water resources are in short supply, and the evaporation process typically represents the ineffective depletion of water resources, thus constituting a crucial aspect in water cycle research. In the Korla region, the hydrogen and oxygen stable isotopes, δD and δ¹⁸O, in groundwater are distributed along the evaporation line, suggesting a pronounced evaporation process during the regional water cycle. In this work, laser isotope spectrometry was utilized to measure the isotopic abundances of various water bodies within the Kaidu–Kongque River Basin. The characteristics of hydrogen and oxygen stable isotopes were analyzed, the evaporation fractionation process was inverted, and a comprehensive systematic analysis was carried out in conjunction with the basin’s water cycle. The results indicate that the mountainous upper Kaidu–Kongque River Basin serves as the groundwater recharge zone. The fitted line for δD and δ¹⁸O in water bodies is δD = 8.67 × δ¹⁸O + 24.01, demonstrating a distribution pattern along the meteoric water line. In the midstream Yanqi Basin, the fitted line for δD and δ¹⁸O in groundwater is δD = 8.18 × δ¹⁸O + 18.35, also showing a distribution along the meteoric water line. The spatial distribution differences are governed by the fractionation effect during the precipitation process. As precipitation clouds move from east to west, δD and δ¹⁸O gradually become depleted. Bosten Lake, situated in the midstream, serves as the discharge terminus of the Yanqi Basin and the sole water source for the downstream Kongque River. It is also the primary site for surface water evaporation within the Kaidu–Kongque River Basin. The inversion of isotopic evaporation fractionation in the lake water reveals that the evaporation ratio can reach 43.4% ± 5.1%. The groundwater in the downstream Korla region is predominantly recharged by the Kongque River water. The fitted line for δD and δ¹⁸O is δD = 4.58 × δ¹⁸O – 19.91, exhibiting characteristics of distribution along the evaporation line. The inversion calculation of evaporation fractionation indicates an average evaporation ratio of 18.3% ± 2.3%. This distribution characteristic is clearly inherited from the evaporation fractionation effect in the midstream Bosten Lake. Given the relatively large groundwater depth in the Korla region, the likelihood of large-scale surface water evaporation during the runoff process can be excluded.