A one-step facile method of synthesizing fluorescent carbon dots (CDs) has been demonstrated, whereby fluorescent CDs are produced through hydrothermal treatment of glucose in the presence of H3BO3 with a fluorescence quantum yield of 14.5%. It is found that spherical CDs have an average size of 3.7[Formula: see text]nm as well as good monodispersion in aqueous solution. The added Cr (VI) selectively leads to the inner filter effect (IFE)-based fluorescence quenching of the CDs. Such fluorescence responses can be used for well quantifying Cr (VI) in the range of 0.05–200[Formula: see text][Formula: see text]M. Significantly, the CDs possess negligible cytotoxicity, excellent biocompatibility and high selectivity. All these features are favorable for label-free monitoring of Cr (VI) in complex biological samples. It was then successfully applied for the fluorescence imaging of intracellular Cr (VI). As an efficient chemosensor, the CDs hold great promise in broadening their applications in biological systems.