A turn-on fluorescent probe, containing a naphthalimide platform and benzothiazole modified by the 2,4-dinitrobenzenesulfonyl (DNBS) group, was designed as an efficient fluorescent probe for sensitive detection of biological thiols and H₂S species regulated by a photoinduced electron transfer (PET) mechanism. This probe shows a speedy response to analytes and has the ability to detect thiols and H₂S in aqueous and living organisms via an obvious turn-on fluorescence response.