The limited availability of uranium (U) resources poses significant challenges to the advancement of nuclear energy. Recycling uranium from spent fuel is critical, but the coexistence of lanthanides (Ln) complicates the extraction process significantly. Here, we present an N/O ligand, (E)-N'-(pyridin-2-ylmethylene) picolinohydrazide (PYPH), designed for the selective recovery of U(VI) over Ln(III/IV) in acidic environments. 3,6-Bis(2-pyridinyl)-1,2,4,5-tetrazine (BPTZ) and N,N-dimethylformamide (DMF), when subjected to heat, gradually generate PYPH and formic acid in aqueous solution; this process can be employed for uranium recovery. This approach, known as the in situ reactive extraction technique, enhances capture capacity, selectivity, and acid resistance while effectively mitigating interference from Ce(IV). At pH 3 and 0.1 M HNO3, separation factors for the binary Ln(III/IV) and U(VI) systems exceeded 103 and 102, respectively, achieving purities exceeding 99%. Monocrystalline structure analysis revealed two-dimensional planar coordination complexes, demonstrating their exceptional selectivity. This study underscores the potential of PYPH in uranium recovery from spent fuel and proposes new avenues for developing innovative separation strategies for lanthanides and actinides (An) using structural and theoretical modeling.