A simple and label-free DNA electrochemical impedance biosensor has been developed for the sequence-specific recognition of double-stranded DNA (ds-DNA). The probe triplex-forming oligonucleotide (TFO) is immobilized on the gold electrode through Au–S interaction. In the presence of the target ds-DNA, the probe TFO interacted with the bases in the major groove of the ds-DNA to form a rigid triplex structure via Hoogsteen hydrogen bonds, leading to an increase of the electron-transfer resistance. It is found that the change of impedance has a linear relationship with the concentration of the target in the range of 0.1–40 nmol L⁻¹, and the detection limit is as low as 0.04 nmol L⁻¹ (S/N = 3).