Understanding the in vivo behavior of experimental therapeutic cells is fundamental to their successful development and clinical translation. Iodine-124 has the longest half-life (4.2 days) among the clinically used positron emitters. Consequently, this isotope offers the longest possible tracking time for directly labeled cells using positron emission tomography (PET). Herein, we have radiosynthesized and evaluated two iodine-124/fluorescein-based dual PET and fluorescent labeling reagents, namely 124I-FIT-Mal and 124I-FIT-(PhS)2Mal for cell surface thiol bioconjugation. 124I-FIT-(PhS)2Mal labeled cells significantly more effectively than 124I-FIT-Mal. It conjugated to various cell lines in 22%–62% labeling efficiencies with prolonged iodine-124 retention. 124I-FIT-(PhS)2Mal mainly conjugated on the cell membrane, which was confirmed by high-resolution fluorescence imaging. The migration of 124I-FIT-(PhS)2Mal labeled Jurkat cells was visualized in NSG mice with excellent target-to-background contrast using PET/CT over 7 days. These data demonstrate that 124I-FIT-(PhS)2Mal can dynamically track cell migration in vivo using PET/CT over a clinically relevant time frame.