Abstract: To address the challenge of evaluating groundwater remediation effectiveness after in situ Leaching (ISL) uranium mining, this study proposes a novel quantitative assessment method based on uranium isotopic fractionation (δ²³⁸U). Taking a long-term suspended CO₂+O₂ ISL mining area in northern China as the research subject, systematic monitoring of δ²³⁸U values, hydrochemical parameters, and uranium concentrations in groundwater samples was conducted, revealing key geochemical behaviors during natural remediation. Preliminary exploration found that the ²³⁸U/²³⁵U fractionation factor ε in the study area is (0.46±0.07)‰, with most monitoring wells showing significantly negative δ²³⁸U values (minimum −2.49‰), indicating the occurrence of U(VI) reduction as the dominant remediation mechanism. Further research demonstrates that δ²³⁸U exhibits a significant negative correlation with redox potential (Eh) and serves as a more reliable direct indicator of U(VI) reduction extent than Eh alone. However, high concentrations of carbonate complexes can inhibit the reduction process, resulting in less negative δ²³⁸U values at strongly reducing sites (Eh < 0 mV). This study confirms that δ²³⁸U can serve as an effective tracer for assessing the efficacy and stability of natural groundwater remediation in ISL mining areas, providing a scientific basis for setting remediation goals and optimizing technical strategies.