A label-free, ultra-sensitive and turn-off fluorescence method for detecting cysteine (Cys) has been developed using enhanced DNA-templated silver nanoclusters (DNA-AgNCs) as the fluorescence probe. The method is based on the specific interaction between Cys and DNA-AgNCs via robust Ag-S bonds and the fluorescence quenching ability of Cys to DNA-AgNCs. Using this method for Cys assay, it was found that the change of fluorescent intensity has a good linear relationship with Cys concentration in the range from 0.1 nM to 100 nM (R2 = 0.991). The detection limit of Cys was 0.05 nM. Furthermore, the method was successfully used for the detection of Cys in human serums, the result of which was confirmed using clinical colorimetric assay. In summary, our study shows that this simple, sensitive and rapid detection method can be hopefully used for theranostics.