The real-time conformational changes of cytosine (C)-rich ssDNA oligonucleotides upon binding with silver ions (Ag(+)) were studied using dual polarization interferometry (DPI). Upon the addition of Ag(+), Ag(+) selectively bound to cytosine-cytosine mismatches and formed C-Ag(+)-C complexes, inducing change of the structure of the C-rich ssDNA from random coil conformation to duplex conformation, whereas the control ssDNA without cytosine-cytosine mismatches had no such signal, which was consistent with circular dichroism (CD) characterization. The conformational change of DNA was reflected on the changes of the mass, thickness, and density values resolved by DPI. The calibration curves showed that as the concentration of Ag(+) increased from 10 nM to 8 μM, the thickness and mass values increased linearly while the density values decreased linearly. Other metal ions such as K(+), Ca(2+), Na(+), Mg(2+), Zn(2+), Mn(2+), Ni(2+), and Pb(2+) did not interfere with the interaction between Ag(+) and C-rich ssDNA, indicating that this method had a good selectivity. The practical application of this biosensor was also investigated in real samples such as drinking water. Besides, cysteine could specifically capture Ag(+) from C-Ag(+)-C complexes and transformed the structure of the C-rich DNA back from rigid double-stranded conformation to random coil conformation, which allowed cysteine to be detected selectively as well. It is expected that this biosensing strategy may be utilized to study the interaction of DNA with other molecules.