Selective and prior extraction of 99TcO4− ahead of uranium and plutonium separation is a beneficial strategy for the modern nuclear fuel cycle. Herein, a novel DGA-grafting pyridine ligand BisDODGA-DAPy (L1) was tailored for the efficient separation of TcO4− from simulated spent nuclear fuel based on the selectivity of pyridine and synergistic effect of diglycolamide (DGA) group. Compared to the ligands BisDOSCA-DAPy (L2) and BisDODGA-MPDA (L3) with similar structure, BisDODGA-DAPy (L1) demonstrated the better separation performance including good extraction efficiency, reusability, and high loading capacity for TcO4− under high acidic medium. The interactions of the ligands with Tc(VII)/Re(VII) have been investigated in detail using FT-IR, 1H NMR titration, UV-Vis spectrophotometric titration, ESI-HRMS and DFT simulations. The extraction mechanism affected by the protonation of ligand was elucidated under different acidity. BisDODGA-DAPy (L1) demonstrated the ultra-selective extraction ability for TcO4− from simulated spent nuclear fuel. The maximum SFTc/U and SFTc/Pu values were up to 1.29×104 and 5.08×103, respectively. In the presence of 9×104-fold excess of NO3−, the extraction of TcO4− was almost unaffected. Moreover, the good radiolytic stability further highlights the promising potential of this ligand for 99Tc separation. DFT calculation revealed the dominant role of DAPy and DODGA in TcO4− extraction, providing the theoretical evidence for BisDODGA-DAPy (L1) to selectively bind TcO4− over NO3−.