Here we report a reusable DNA single-walled carbon nanotube (SWNT)-based fluorescent sensor for highly sensitive and selective detection of Ag(+) and cysteine (Cys) in aqueous solution. SWNTs can effectively quench the fluorescence of dye-labeled single-stranded DNA due to their strong pi-pi stacking interactions. However, upon incubation with Ag(+), Ag(+) can induce stable duplex formation mediated by C-Ag(+)-C (C=cytosine) coordination chemistry, which has been further confirmed by DNA melting studies. This weakens the interactions between DNA and SWNTs, and thus activates the sensor fluorescence. On the other hand, because Cys is a strong Ag(+) binder, it can remove Ag(+) from C-Ag(+)-C base pairs and deactivates the sensor fluorescence by rewrapping the dye-labeled oligonucleotides around the SWNT. In this way, the fluorescence signal-on and signal-off of a DNA/SWNT sensor can be used to detect aqueous Ag(+) and Cys, respectively. This sensing platform exhibits high sensitivity and selectivity toward Ag(+) and Cys versus other metal ions and the other 19 natural amino acids, with a limit of detection of 1 nM for Ag(+) and 9.5 nM for Cys. Based on these results, we have constructed a reusable fluorescent sensor by using the covalent-linked SWNT-DNA conjugates according to the same sensing mechanism. There is no report on the use of SWNT-DNA assays for the detection of Ag(+) and Cys. This assay is simple, effective, and reusable, and can in principle be used to detect other metal ions by substituting C-C base pairs with other native or artificial bases that selectively bind to other metal ions.