Electrochemical impedance spectroscopy study of electroactive poly(epsilon-caprolactone)/polypyrrole (PCL/PPy) electrospun nanofibers which are functionalized with single-stranded DNA (ssDNA) were performed for the first 01 time. PCL nanofibers which have high loading capacity for bioactive molecules were fabricated by electrospinning. ssDNA were immobilized onto PCL/PPy nanofibers following to pyrrole (Py) polymerization on electrospun PCL nanofibers. Electrochemical impedance spectroscopy (EIS) and equivalent electrical circuit modeling was used to investigate the changes in interfacial electrical properties of PCL/PPy nanofibers that arise from ssDNA immobilization, and hybridization with complementary and non-complementary DNA sequences. The surface morphology of the DNA immobilized PCL/PPy nanofibers become rough and the fiber diameters were increased. Elemental analyses and EDX-mapping showed that the ssDNA was evenly distributed on the surface of the nanofibers. Charge transfer resistance (Rct) of ssDNA/PPy/PCL nanofibers were decreased with the increasing concentrations of immobilized DNA. After hybridization with fully complementary DNA, significant decreased in charge transfer resistance and solution resistance was observed due to change of surface charge of nanofibers and conformation of DNA during the hybridization process. PCL/PPy nanofiber mats were found to be promising materials which would be used in electrochemical biosensor applications.