A novel gold nanoparticle (AuNP)-based sensor for detecting thiols in aqueous solution has been developed. Due to the weak N···Au interactions, meso-(4-pyridinyl)-substituted BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes were coordinated to AuNP surfaces, which effectively quenched the fluorescence of organic/inorganic hybrid systems. The fluorescent quenching mechanism was mainly ascribed to the highly efficient fluorescent resonance energy transfer (FRET) and the inner filter effect. In the presence of thiols, meso-(4-pyridinyl)-substituted BODIPY chromophore were displaced and released from the AuNP surfaces and thus restored the fluorescence of BODIPY chromophore. The modulation of the fluorescence quenching efficiency of BODIPY–AuNPs in the presence of thiols can achieve a large turn-on fluorescence enhancement (40-fold) in aqueous solution. The new AuNP-based fluorescence sensor displayed desired properties such as high specificity, relatively low detection limit (30 nM for Cys), appreciable water solubility and rapid response time (within 2 min for Cys/Hcy). Moreover, the sensor has been successfully applied for monitoring and imaging of intracellular thiols within living HeLa cells.