Water-soluble conjugated polymers (CPS) are a versatile class of advanced organic materials with excellent photochemistry properties. Using a biotin-modified fluorescence quencher (quencher-tether-ligand, QTL) to specifically sense the presence of avidin based on CP is of great importance. However, the nonspecific interactions between various proteins and Cps greatly baffle the studies of the mechanism and applications of CP-based biosensors. We selected luminescent ruthenium(II) polypyridine biotin complex (Ru-biotin) as a proper probe, which can not only work as a fluorescent quencher but also produces fluorescence resonance energy transfer (FRET) with anionic water-soluble CP (poly [5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenecinylene] (MPS-PPV)). As the emission peak of Ru-biotin produced by FRET cannot be infected by nonspecific proteins, we discussed the MPS-PPV/Ru-biotin/avidin biosensing mechanism based on the fluorescence spectra changes of polymer and Ru-biotin and consequently realized the rapid and specific detection of avidin.