Semisynthetic fluorescent biosensors consisting of a protein framework and a synthetic fluorophore are powerful analytical tools for specific detection of biologically relevant molecules. We report herein a novel method that allows for the construction of turn-on fluorescent semisynthetic biosensors in a one-step manner. The strategy is based on the ligand-directed tosyl (LDT) chemistry, a new type of affinity-guided protein labeling scheme which can site-specifically introduce synthetic probes to the surface of proteins with concomitant release of the affinity ligands. Novel quenched ligand-directed tosylate (Q-LDT) reagents were designed by connecting an organic dye to a conjugate of a protein ligand and a fluorescence quencher through a tosyl linker. The Q-LDT-mediated labeling directly converts a natural protein to a fluorescently labeled protein that remains noncovalently complexed with the cleaved ligand-tethered quencher. The fluorescence of this labeled protein is initially quenched and only in the presence of specific analytes is the fluorescence enhanced (turned on) due to the expulsion of the ligand-quencher fragment. Using a single labeling step, this approach was successfully applied to carbonic anhydrase II (CAII) and a Src homology 2 (SH2) domain to generate turn-on fluorescent biosensors toward CAII inhibitors and phosphotyrosine peptides, respectively. Detailed investigations revealed that the obtained biosensors exhibit their natural ligand selectivity. The high target-specificity of the LDT chemistry also allowed us to prepare the SH2 domain-based biosensor not only in a purified form but also in a bacterial cell lysate. These results demonstrate the utility of the Q-LDT-based approach to expand the applications of semisynthetic biosensors.