Abnormal levels of intracellular biothiol species, including glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), are closely relevant to a variety of diseases. In this study, we first report the design and synthesis of a 3-thiolflavone-based probe 3-STF with an S-S linkage both as the fluorescence quenching agent and the recognition site. Upon treatment with biothiols, the S-S linkage was cleaved by nucleophilic attack of RS, and 3-STF exhibited a specific "turn-on" fluorescence response at 543.8 nm upon 390 nm excitation. Meanwhile, 3-STF was proved to be highly sensitive and selective for the detection of biothiols over other nucleophiles such as amino acids and HS. The sensing mechanism was further verified by H NMR and ESI-MS analysis. In addition, 3-STF with low cytotoxicity can be successfully applied to detect endogenous and exogenous biothiols in HepG-2 cells.