The competition of uranium and vanadium ions is a major challenge in extracting uranium from seawater. In-depth exploration of the complexation of uranium and vanadium ions with promising ligands is essential to design highly efficient ligands for selective recovery of uranium. In this work, we systematically explored the uranyl and vanadium extraction complexes with three tetradentate N,O-mixed donor analogues including the rigid backbone ligands 1,10-phenanthroline-2,9-dicarboxylic acid (PDA, L¹) and 5H-cyclopenta[2,1-b:3,4-b′]dipyridine-2,8-dicarboxylate acid (L³), as well as the flexible ligand [2,2′-bipyridine]-6,6′-dicarboxylate acid (L²) using density functional theory (DFT). These ligands coordinate to the uranyl cation in a tetradentate fashion, while L¹ and L³ act as tridentate ligands toward VO₂⁺ due to the smaller ionic radius of VO₂⁺ and larger cleft sizes of L¹ and L³. Bonding analyses show that the metal–ligand bonding orbitals of the uranyl complexes [UO₂L(CO₃)]²⁻, [UO₂L(OH)]⁻, and [UO₂L(H₂O)] mainly arise from the interactions of the U 5f, 6d orbitals and N, O 2p orbitals. Because of the rigid structure and more suitable chelate ring size, the L¹ ligand possesses a stronger complexing ability for uranyl ions than other ligands, while the L³ ligand has weaker binding affinity than L¹ and L². All these ligands prefer to coordinate with the uranyl cation rather than vanadium ion, indicating the selectivity of these ligands to [UO₂(CO₃)₃]⁴⁻ over H₂VO₄⁻ and HVO₄²⁻ in seawater. This is mainly attributed to the metal ion size-based selectivity and structural preorganization of the ligands. These results demonstrate that the backbone of these ligands affect their extraction behaviors. It is expected that this work might prove useful in designing efficient ligands for uranium extraction from seawater.