We report the synthesis and characterization of luminescent rhenium(I) amidodipyridoquinoxaline biotin complexes [Re(CO)(3)(dpqa)(L)](PF6) (dpqa = 2-(n-butylamido)dipyrido[3,2-t.2',3'-h]quinoxaline; L = 4-(biotinamidomethyl)pyridine (py-4-CH2-NH-biotin) (1), 3-(N-((2-biotinamido)ethyl)amido)pyridine (py-3-CO-NH-en-NH-biotin) (2), 4-(N-((6-biotinamido)hexanoyl)aminomethyl)pyridine (py-4-CH2-NH-cap-NH-biotin) (3)), and their biotin-free counterpart [Re(CO)(3)(dpqa)(py)](PF6) (py = pyridine (4)). Upon irradiation, these complexes exhibited intense triplet metal-to-ligand charge-transfer ((MLCT)-M-3) (d pi(Re) -> pi*(dpqa)) emission in fluid solutions at 298 K and in alcohol glass at 77 K. However, the emission became much weaker in aqueous buffer, probably due to the interactions of water molecules with the amide substituent of the dpqa ligand. These properties render the complexes good candidates as luminescent probes for hydrophobic media, such as the substrate-binding sites of proteins. The avidin-binding properties of the new biotin complexes have been studied by 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assays, emission titrations, and competitive association and dissociation assays. Most importantly, the complexes showed a profound increase in emission intensities upon binding to avidin. Additionally, we found that the fluorescence of anthracene was quenched by these rhenium(I) complexes, and the (MLCT)-M-3 emission of the complexes was also quenched by anthracene. On the basis of these findings, new homogeneous assays for biotin using these complexes, avidin, and anthracene-labeled avidin have been designed.