We describe in this report a development of label-free electrochemical DNA sensor based on a novel nanostructured electrode of multi-walled carbon nanotubes (MWCNTs)/ nano-flowers-like manganese dioxide (MnO2)/polyaniline nanowires (PANi NWs) nanocomposite. The nanocomposite was synthesized in-situ onto an interdigitated platinum microelectrode (Pt) using a combination of chemical and electrochemical synthesis methods: chemical preparation of MWCNTs/MnO2 and electropolymerization of PANi NWs. The fabricated MWCNTs/MnO2/PANi NWs was then used to develop a label-free electrochemical DNA sensor for a specific gene of Escherichia coli (E.coli) O157:H7 detection. The MWCNTs/MnO2/PANi NWs modified Pt electrode’s surface can facilitate for probe DNA strands immobilization and, therefore the electrochemical signal of the DNA sensors has been improved. The electrochemical impedance spectroscopy (EIS) measurements were conducted to investigate the output signals generated by the specific binding of probe and target DNA sequences. Obtained results indicated that the developed electrochemical biosensor can detect the target DNA in the linear range of 5 pM to 500 nM with a low limit of detection (LOD) at 4.42 × 10 –13 M. The research results demonstrated that the MWCNTs/MnO2/PANi NWs nanocomposite-based electrochemical DNA sensor has a great potential application to the development of highly sensitive and selective electrochemical DNA sensors to detect pathogenic agents.