We report a label-free fluorescence turn-on approach for the sensitive and selective sensing of silver ion Ag(+) based on Ag(+) induced conformational change of cytosine-rich single-stranded DNA. A silver specific oligonucleotide (SSO) undergoes a structural transition from single-stranded DNA (ssDNA) to a C-Ag(+)-C mediated hairpin structure, which can be recognized by a double-stranded DNA (dsDNA) intercalator Sybr Green I (SG). The linear response range for Ag(+) detection was from 1 nM to 100 nM. The method presented here is highly sensitive and a limit of detection of 1 nM was obtained. More importantly, this optical method was successfully used to identify complex sample mixtures.