Bifunctional chelators play an important role in developing metallic radionuclide-based radiopharmaceuticals. In this study, a new bifunctional ligand, p-SCN-PhPr-NE3TA, was synthesized and conjugated to a very late antigen-4 targeting peptidomimetic, LLP2A, for evaluating its application in Cu-64-based positron emission tomography (PET) imaging. The new ligand exhibited strong selective coordination of Cu(II), leading to a robust Cu complex, even in the presence of 10-fold Fe(III). The LLP2A conjugate of p-SCN-PhPr-NE3TA was prepared and successfully labeled with Cu-64 under mild conditions. The conjugate Cu-64-NE3TA-PEG(4)-LLP2A showed significantly higher specific activity, compared with Cu-64-NOTA-PEG(4)-LLP2A, while maintaining comparable serum stability. Subsequent biodistribution studies and PET imaging in vivo performance and high tumor uptake with low background, chelator for "Cu-based radiopharmaceuticals. in mice bearing B16F10 xenografts confirmed its favorable rendering p-SCN-PhPr-NE3TA a promising bifunctional chelator for Cu-64-based radiopharmaceuticals.