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In this work, a novel 7-hydroxybenzoxazinone-based fluorescent probe (<b>PBD</b>) for the selective sensing of biothiols is reported. Upon treatment with biothiols, <b>PBD</b> shows a strong fluorescence enhancement (up to 70-fold) and a large Stokes shift (155 nm). Meanwhile, this probe exhibits high resistance to interference from other amino acids and competing species. <b>PBD</b> features good linearity ranges with a low detection limit of 14.5 nM for glutathione (GSH), 17.5 nM for cysteine (Cys), and 80.0 nM for homocysteine (Hcy), respectively. Finally, the potential utility of this probe for biothiol sensing in living HeLa cells is demonstrated.