In this work, sulfur and nitrogen co-doped carbon dots (S,N-CQDs) as highly selective fluorescent probe for silver ion (Ag) and cysteine (Cys) detection were designed and synthesized directly from citric acid and thiamine hydrochloride via a one-step hydrothermal protocol in 63.8% quantum yield. This probe enabled selective detection of Ag with a linear range of 0-10 and 10-250μM and a limit of detection of 0.40μM with respect to the variation in fluorescence induced by target concentration and electron-transfer from S,N-CQDs to Ag. Furthermore, S,N-CQDs/Ag fluorescence can be effectively recovered by virtue of a specific reaction of Cys with silver ion. This fluorescence "turn-on" protocol was applied to determine Cys with two linear ranges of 0-10 and 10-120μM as well as a detection limit of 0.35μM. The corresponding cell experiments were also performed, indicating that the prepared S,N-CQDs possessed low cytotoxicity and good biocompatibility. Ultimately, the practicality and viability of this fluorescent probe were demonstrated through the analysis of silver ion in real river water and human serum samples.