Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and involved in cellular uptake of solute. In this study, glutamine binding PBP (GlnBP) and leucine binding PBP (LeuBP) were engineered to detect L-Glu and L-Leu by FRET change upon ligand binding. A fluorescent unnatural amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and emerald green fluorescent protein (EGFP) or yellow fluorescent protein (YFP) was fused to its N-terminus as a FRET acceptor. The best sensor protein showed 1.5-fold increase in FRET ratio. In the case of leucine sensor protein, its substrate specificity was significantly improved by engineering the LeuBP, showing minimal FRET ratio change with the other 19 natural amino acids and D-Leu. Further engineering made the sensor protein more sensitive (14-fold) for L-Leu, and recognize L-Met as well with moderate binding affinity. Selected mutant sensors were used to measure L-Glu and L-Leu concentration in a biological sample (fetal bovine serum) and optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to readily engineer the natural receptor to have improved binding affinity and specificity, and to recognize other natural molecules which are not a ligand for the wild-type receptor. The design strategy can be applied to other natural receptors and would make it possible to engineer the receptors to sense biochemically more interesting molecules.