Autoinducer-2 (AI-2) is a Quorum Sensing (QS) molecule utilized by bacteria in interspecies communication. More recently, it is identified to be vital in regulating QS pathways in a number of human and foodborne pathogens. Methods to detect AI-2 in a rapid and highly sensitive manner can help in the early detection of bacterial infections. Herein, we describe a rapid, selective, and highly sensitive protein based biosensing system employing the Fluorescence Resonance Energy Transfer (FRET) between a protein fusion LuxP-EGFP and 7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin (MDCC). The developed biosensing system, which can detect AI-2 at subnanomolar levels, was successfully applied to detect AI-2 in clinical samples such as saliva and blood serum.