An unlabeled immobilized DNA-based sensor was reported for simultaneous detection of Pb(2+), Ag(+), and Hg(2+) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)(6)](4-/3-) as redox probe, which consisted of three interaction sections: Pb(2+) interaction with G-rich DNA strands to form G-quadruplex, Ag(+) interaction with C-C mismatch to form C-Ag(+)-C complex, and Hg(2+) interaction with T-T mismatch to form T-Hg(2+)-T complex. Circular dichroism (CD) and UV-vis spectra indicated that the interactions between DNA and Pb(2+), Ag(+), or Hg(2+) occurred. Upon DNA interaction with Pb(2+), Ag(+), and Hg(2+), respectively, a decreased charge transfer resistance (R(CT)) was obtained. Taking advantage of the R(CT) difference (ΔR(CT)), Pb(2+), Ag(+), and Hg(2+) were selectively detected with the detection limit of 10 pM, 10 nM, and 0.1 nM, respectively. To simultaneously (or parallel) detect the three metal ions coexisting in a sample, EDTA was applied to mask Pb(2+) and Hg(2+) for detecting Ag(+); cysteine was applied to mask Ag(+) and Hg(2+) for detecting Pb(2+), and the mixture of G-rich and C-rich DNA strands were applied to mask Pb(2+) and Ag(+) for detecting Hg(2+). Finally, the simple and cost-effective sensor could be successfully applied for simultaneously detecting Pb(2+), Ag(+), and Hg(2+) in calf serum and lake water.