Uranium, as a significant radioactive element in nature, demonstrates considerable scientific importance through its isotopic fractionation phenomena in studies of uranium mineralization genesis, geochemical process tracing, and environmental remediation. This contribution systematically synthesizes uranium isotope fractionation mechanisms (nuclear volume effects, redox, adsorption, and leaching processes), advanced analytical methodologies (chemical separation protocols and mass spectrometric techniques), and characteristic isotopic signatures of principal global reservoirs (continental crust, mantle, and marine systems). Furthermore, it examines applications in characterizing typical uranium deposit types, including sandstone- hosted and granite- related mineralization systems. The analysis underscores the distinctive capacity of uranium isotopes in reconstructing metallogenic environments, tracing ore- forming material sources, and deciphering redox evolution. Prospective research directions are proposed regarding deep planetary processes, mineralization mechanisms, and mine remediation monitoring, thereby providing critical insights for advancing genetic models of uranium deposits and enhancing exploration strategies.