Chromium is a toxic metal can do severe damage to public health, plants, and aquatic animals due to its high carcinogenicity. Cr(VI) is about 1000 times more toxic than Cr(III). As a result, exploring methods for differentiation between Cr(III) and Cr(VI) and quantification of these species with high selectivity are essential in aquatic analysis. In this paper, carbon-dots (C-dots) were successfully synthesized with high quantum yield and applied for the determination of the concentration of Cr(VI). It was found that C-dots and Cr (VI) were effective fluorophore and quencher pair for the fluorescence sensor based on inner filter effect. The fluorescence intensity of the C-dots was highly sensitive to the change of concentration of Cr (VI) with good analytical performance. Since ascorbic acid (AA) has been advocated as a treatment of systemic chromium poisoning by converting Cr(VI) to Cr(III), the determination of AA was also investigated. A standard addition methodology was used to determine the Cr(VI) concentration in water samples and the concentration of AA in human serum samples with good results. In summary, compared to traditional methods, the method using C–dots as fluorescence probe is sensitive, selective, cheap and eco-friendly for the determination of the concentration of Cr(VI) and ascorbic acid.