Herein, an electrochemical human immunoglobulin G (HIgG) immunosensor was fabricated using overoxidized polypyrrole decorated with gold nanoparticle (OPPy-Aunano) modified screen printed electrode (OPPy-Aunano/SPE). The results clearly demonstrate that OPPy-Aunano film is very permeable to [Fe(CN)6]3−/4−. Therefore, the fabrication of immunosensor based on permeability of OPPy-Aunano film to [Fe(CN)6]3−/4− probe is a novel strategy for the fabrication of label free electrochemical immunosensor. The electrochemical impedance spectroscopy (EIS) techniques are used for the determination of HIgG in the concentration range 0.5 to 125.0ngmL−1 and with a limit of detection of 0.02ngmL−1, respectively.