In order to enhance the understanding of the characteristics and potential of geothermal resources in the Dunhuang Basin to support exploration and development initiatives, through the application of geothermal exploration techniques and hydrogeochemical analysis, the methods including hydrogen and oxygen isotope testing, Piper diagram analysis, the K-Mg geothermometer, and thermal storage estimation was used to investigate the chemical properties of geothermal water, the recharge source and age, as well as to calculate the thermal storage temperature and geothermal water circulation depth. Furthermore, the potential of geothermal resources in the basin was comprehensively evaluated. The results indicate that the thermal reservoir is primarily composed of argillaceous sandstone, pebbly fine sandstone, and glutenite located in the lower member of the Neogene Shulehe Formation, with the caprock consisting of Quaternary loose rock as well as mudstone and sandy mudstone in the upper member of the same formation. The primary heat source is heat conduction from the upper mantle and deep crust. The geothermal waters are dominated by Cl·SO₄ -Na hydrochemical components, with temperatures ranging between 28.7 ℃ and 38.0 ℃ and total dissolved solids (TDS) values varying from 1 146 mg/L to 3 250 mg/L. Isotopic analysis, including δD, δ¹⁸ O, ³H, and ¹⁴C, reveals that the geothermal water represents a mixture of deep groundwater and modern precipitation. The estimated reservoir temperatures range from 39.49 ℃ to 42.75 ℃, and the calculated circulation depths are between 1 020.65 m and 1 268.34 m. These findings suggest that the geothermal resources in the Dunhuang Basin are derived from deep groundwater circulation, replenished by atmospheric precipitation from the southern mountainous regions. The calculated thermal potential modulus of the geothermal fluid is 1.78 × 10⁹ kJ/(km²·a), indicating significant resource potential for future utilization.