Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.