The development of ideal peptide-based radiopharmaceuticals faces critical bottlenecks, primarily due to limited cellular internalization and insufficient deep tissue penetration of peptide carriers. To address this, we developed an intracellular targeting and DNA-adjacent radiotherapeutic strategy using stapled peptides. The MDM2/MDMX-targeting stapled peptide-based radiopharmaceuticals, denoted as [64Cu]Cu-DOTA-STP, exhibited prolonged circulation in the bloodstream and slow systemic clearance. Furthermore, in vitro studies demonstrated that nearly 50% of administered [64Cu]Cu-DOTA-STP was internalized, achieving efficient intracellular accumulation. In addition, [64Cu]Cu-DOTA-STP demonstrated high tumor accumulation, with a standard uptake value of up to 9.39 ± 1.52%ID/g. Finally, targeted radionuclide therapy confirmed that [64Cu]Cu-DOTA-STP effectively inhibited tumor growth, irrespective of p53 phenotypes. Taken together, this study leveraged PET imaging as a noninvasive and longitudinal tool to elucidate the in vivo fate of stapled peptides and demonstrated that stapled peptides can serve as ideal vehicles for developing intracellular protein-targeting radiopharmaceuticals, achieving efficient 64Cu-based targeted radionuclide therapy.