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A highly effective strategy for quantification of plasmid which was a special dsDNA based on bisPNA by electrochemical impedance spectroscopy was presented in this work. Firstly, through Au−S bond, thiol‐terminated bisPNA probes were immobilized onto the gold electrode surface. Then bisPNA probes directly hybridized with target plasmid DNA pBR322 based on the PNA.DNA‐PNA invasion triplex without denaturation. In the presence of redox electroactive ions [Fe(CN)6]3−/4− as hybridization indicator, the charge transfer resistance (Rct) was produced, and Rct was measured via electrochemical impedance spectroscopy. Under optimal conditions, this strategy showed a good linear relationship between the ΔRct which was the difference of Rct obtained before and after bisPNA hybridized with plasmid pBR322, and logarithm of the concentration of plasmid pBR322 within the range from 1 nM to 100 nM (R2=0.993), with a limit of detection (LOD) of 0.1 nM. Furthermore, this bisPNA‐assisted biosensor showed good stability and satisfactory analytical reliability. In addition, this novel bisPNA‐assisted biosensor also exhibited excellent analytical results in human serum. [ABSTRACT FROM AUTHOR]