The successful development of targeted nanoparticle (NP)-based therapeutics depends on the effective conjugation of targeting ligands to the NP. However, conventional methods based on chemical reactive groups such as N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyi) carbodiimide, and maleimide have several limitations, including low binding efficiency, complex reaction methods, long reaction times, and reduced activity of the targeting ligand. In this study, we developed a novel method for conjugating targeting ligands to albumin NPs using the recently developed bacterial superglue the SpyTag/SpyCatcher (ST/SC) ligation system. This method involves a rapid one-step conjugation process with almost 100% efficiency. Albumin NPs conjugated to human epidermal growth factor receptor 2 (HER2) affibody molecules using the ST/SC system showed strong binding to HER2-overexpressing cells. In addition, NPs encapsulated with indocyanine green accumulated in cells overexpressing HER2 and exhibited superior photothermal treatment effects. Thus, surface functionalization of NPs using the ST/SC reaction may be used to develop new nanosystems that exhibit improved therapeutic benefits.