Conformational switch from hairpin DNA to G-quadruplex induced by Pb²⁺ is studied by electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)₆]^3−/4− as the redox probe. In the presence of Pb²⁺, the G-rich hairpin DNA opens the stem-loop and forms G-quadruplex structure, which gives rise to a sharp increase in the charge-transfer resistance (R_CT) of the film reflected by the EIS. This structural change is also confirmed by circular dichroism (CD) measurements and UV-Vis spectroscopic analysis and calculated by density functional theory (DFT). On the basis of this, we develop a label-free electrochemical DNA biosensor for Pb²⁺ detection. With increasing concentrations of Pb²⁺, the differences in the charge-transfer resistance R_CT before and after the Pb²⁺ incubation is linearly dependent on the logarithm of Pb²⁺ concentration within a range from 50 μM to 0.5 nM. The biosensor also exhibits good selectivity for Pb²⁺ over other metal ions. This is a simple and label-free electrochemical method for Pb²⁺ detection making use of the G-quadruplex.