Abstract: Medical radioactive isotopes play a very important role in disease diagnosis and treatment, with ⁹⁹Tc^m drugs being widely used in disease diagnosis. ⁹⁹Tc^m is primarily obtained from its parent nuclide, ⁹⁹Mo, through a ⁹⁹Mo/⁹⁹Tc^m generator. Currently, the main methods of producing ⁹⁹Mo include reactor production, accelerator-based production, and neutron generator production. Among these, the fission of high-enriched uranium(HEU) or low-enriched uranium(LEU) targets in reactors is the predominant method for producing ⁹⁹Mo. The dissolved target solution contains large amounts of uranium and fission products, and the similar chemical properties of Mo(Ⅵ) and U(Ⅵ) make their separation particularly challenging. Therefore, effective separation of Mo(Ⅵ) from U(Ⅵ) is crucial for obtaining high-purity ⁹⁹Mo. Solvent extraction, compared to other separation methods, offers advantages such as relatively simple operation and high separation efficiency. The key to liquid-liquid extraction lies in designing efficient ligands to achieve effective separation of Mo(Ⅵ)/U(Ⅵ). In this study, three unilateral derivatized phenanthroline amide ligands were synthesized, and extraction experiments were conducted at different nitric acid concentrations, times and ligand concentrations to comprehensively investigate the separation performance of the three ligands for Mo(Ⅵ)/U(Ⅵ). Coordination chemistry was studied through fourier transform infrared spectroscopy(FTIR), ultra violet visible spectroscopy(UV-Vis) titration and nuclear magnetic resonance spectroscopy(NMR) titration. The experimental results indicate that the distribution ratios of Mo(Ⅵ) and U(Ⅵ) for the three ligands decrease with increasing acidity. This is mainly due to the absence of side chains on one side of the ligand, making the nitrogen atoms on the phenanthroline moiety more prone to protonation. All three ligands show a higher affinity for Mo(Ⅵ), enabling efficient separation of Mo(Ⅵ)/U(Ⅵ) at low acidity. Under optimal conditions, the separation factor(SF(Mo/U)) reaches 23. The three unilateral derivatized phenanthroline amide ligands show the same extraction trend for Mo(Ⅵ)/U(Ⅵ), primarily because they have the same ligand backbone, differing only in their alkyl chains, which do not participate in coordination. Unilateral derivatized phenanthroline amide ligands show faster kinetics compared to phenanthroline diamide ligands, mainly due to their fewer side chains and smaller steric hindrance, which are more conducive to coordination. Since the three ligands are tridentate ligands, they readily occupy the equatorial coordination sites of uranyl and molybdenyl ions. However, the presence of two oxygen atoms along the vertical axis prevents further coordination by the ligands, thus precluding the formation of 2∶1 complexes between the ligand and uranyl or molybdenyl ions. The slope analysis fitting indicates the formation of a 1∶1 complex between the ligand and Mo(Ⅵ)/U(Ⅵ). UV-Vis titration analysis and NMR titration analysis confirm the formation of a 1∶1 complex between the ligand and U(Ⅵ).