Food is one of the most important areas in human health worldwide. Staphylococcus aureus is a bacterium, which cause foodborne diseases. Conductive polymers belong to an interesting group of polymers, which have unique electrical, electrochemical and optical properties. Polyaniline (PANI) possesses exceptional properties like structural, thermal and electrical. In the present work, PANI films were deposited by using electrochemical technique on ITO substrate with different segment cycles. The structure and morphology of PANI has been determined by using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM), respectively. The diffraction pattern of pure PANI showed peaks at 25.5°, which are characteristic peaks of PANI. SEM images of PANI films deposited with less segment cycle showed only agglomerates and particulates. With increase in segment cycle films showed relatively uniform nanofibers with porous nature which is best suited for sensor applications. The FTIR studies revealed the formation of PANI. The transmission peaks of PANI at 2920 cm-1, 2853 cm-1 corresponds to N-H characteristic band present in aromatic amines. Optical absorption peak was observed at 362 nm indicating principal absorption of the π-π∗ orbital transitions of the emeraldine form of PANI. The fabricated voltammetric based sensor showed rapid detection of Staphylococcus aureus microbes at room temperature. Copyright © 2017 American Scientific Publishers All rights reserved.