question: We herein describe a novel and efficient method for the detection of protein/small molecule (SM) interactions, which relies on the protein-induced fluorescence enhancement (PIFE). In this method, a duplex probe is designed to position Cy3 and SM at the optimal distance to maximize the effect of PIFE, which is utilized as the key component. In the presence of target proteins that bind to SM, the Cy3 is guided close to the target proteins, which significantly enhances the fluorescence signal through a process of PIFE. With this approach, we successfully analyzed a model target protein, streptavidin (STV) that interacts with biotin (BTN) in less than 10 min without any washing steps. In addition, the practical applicability of this method was demonstrated by reliably determining STV in human serum. Finally, the universal applicability of this method was demonstrated by monitoring the interaction between folate and folate receptors.