Fluorescent carbon dots (CDs) have been identified as potential nanosensors and attracted tremendous research interests in wide areas including anti-counterfeiting, environmental and biological sensing and imaging in considering of the attractive optical properties. In this work, we present a CDs based fluorescent sensor from polyvinylpyrrolidone, citric acid, and methionine as precursors by hydrothermal approach. The selective quantifying of Fe³⁺ and ascorbic acid (AA) are based on the fluorescent on–off–on process, in which the fluorescent quenching is induced by the coordination of the Fe³⁺ on the surface of the CDs, while the fluorescence recovery is mainly attributed to redox reaction between Fe³⁺ and AA, breaking the coordination and bringing the fluorescence back. Inspired by the good water solubility and biocompatibility, significant photostability, superior photobleaching resistance as well as high selectivity, sensitivity, and interference immunity, which are constructed mainly from the N,S-doping and methionine surface functionalization, the CDs have not only been employed as fluorescence ink in multiple anti-counterfeiting printing and confidential document writing or transmitting, but also been developed as promising fluorescence sensors in solution and solid by CDs doped test strips and hydrogels for effectively monitoring and removing of Fe³⁺ and AA in environmental aqueous solution. The CDs have been also implemented as effective diagnostic candidates for imaging and tracking of Fe³⁺ and AA in living cells, accelerating the understanding of their function and importance in related biological processes for the prevention and treatment specific diseases.