Background <p>Although molecular tumor-targeting peptides modified with albumin-binding domains can enhance tumor probe uptake and prolong tumor retention time, the prolonged systemic circulation resulting from this modification also increases radiation exposure to normal tissues. This study aims to validate that peptide nucleic acid (PNA)-mediated pretargeted ¹⁶¹Tb radionuclide therapy can slow tumor growth and reduce systemic radiation exposure.</p> Results <p>⁶⁸Ga-EB-RGD, ⁶⁸Ga-cPNA-PNA-EB-RGD, ¹⁶¹Tb-EB-RGD, and ¹⁶¹Tb-cPNA-PNA-EB-RGD all demonstrated labeling efficiencies exceeding 80% and exhibited high stability in phosphate-buffered saline. ¹⁶¹Tb-EB-RGD and ¹⁶¹Tb-cPNA-PNA-EB-RGD showed comparable tumor cell uptake and apoptosis induction (0.95-fold). The binding capability between the pretargeting receptor PNA-EB-RGD and the ligand cPNA reached 80%. The blood biodistribution of the pretargeting group (¹⁶¹Tb-cPNA-PNA-EB-RGD) was 32-fold lower than that of the conventional group (¹⁶¹Tb-EB-RGD) (0.37 ± 0.05%ID/g vs. 11.93 ± 0.62%ID/g, <i>P</i> &lt; 0.05). The biodistribution of ¹⁶¹Tb-EB-RGD and ¹⁶¹Tb-cPNA-PNA-EB-RGD in 4T1 tumor cells was 8.77 ± 0.16%ID/g and 3.86 ± 0.21%ID/g, respectively. The conventional treatment group (¹⁶¹Tb-EB-RGD) showed superior therapeutic efficacy compared to the pretargeting group (¹⁶¹Tb-cPNA-PNA-EB-RGD), which in turn outperformed the control group (345.09 ± 4.93 vs. 100.76 ± 12.20 vs. 183.02 ± 12.94 mm³, <i>P</i> &lt; 0.05). No significant pathological changes were detected in the vital organs of treated animals. Ex vivo tumor analysis supported the treatment trends.</p> Conclusions <p>Compared to conventional non-pretargeted radionuclide therapy, PNA-based pretargeted ¹⁶¹Tb radionuclide therapy reduced systemic radiation exposure, significantly decreased hematotoxicity, and suppressed tumor growth in tumor-bearing mice.</p>