Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a without its denaturation using a gold electrode is described. The Electrochemical DNA sensor relies on the modification of gold electrode with 6-mercapto-1-hexanol and self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principal of the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.84 × 10−12 M, in phosphate buffer solution, pH 7.0. The relative standard deviation over three independently probe modified electrodes measured at 100 pM of target ds-DNA was 3.1 % indicating a remarkable reproducibility of the detection method.