Radioactive isotope labeled gold nanomaterials have potential biomedical imaging applications. Here, we report the synthesis and characterization of PEGylated crushed gold shell-radioactive iodide-124 labeled gold core nanoballs (PEG-124I-Au@AuCBs) for in vivo biomedical imaging application through combined positron emission tomography and cerenkov luminescent imaging (PET/CLI). PEG-124I-Au@AuCBs showed high stability and sensitivity in various pH solutions, serum, and in vivo conditions and were not toxic to tested cells. Combined PET/CLI clearly revealed disease lesions at 1 to 24 h after injection of particles, consistent with the biodistribution results. Taken together, the data provided strong evidence for the application of PEG-124I-Au@AuCBs as promising imaging agents in nuclear medicine imaging of various biological systems, particularly in diagnostics of various disease.