Iron oxide nanoparticles (IONPs) have shown great promise in targeted cancer therapy due to their unique magnetic properties and ability to be functionalized with various ligands. This study explores the use of iron oxide nanoparticles (IONPs) functionalized with macrocycle antagonists to target CXCR4 receptors on cancer cells. The synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) was validated through XRD and TEM analyses, which showed uniform, roughly spherical particles. Fluorescence-loaded SPIONs provided enhanced imaging contrast in Jurkat cancer cells. Flow cytometry demonstrated that the nanoparticles effectively blocked CXCR4 receptors, highlighting their potential for targeted cancer therapy. These findings underscore the successful synthesis, characterization, and functionalization of SPIONs, paving the way for advanced nanomedicine strategies in cancer diagnostics and treatment.