Patients with coeliac disease, a gluten intolerance autoimmune disorder, have elevated levels of antitransglutaminase antibody in their human sera. In this study, an immunosensor for the electrochemical determination of antitransglutaminase antibody was constructed. The immunosensor architecture was based on the electrostatic deposition of transglutaminase antigen on a glassy carbon electrode modified with electrosynthetic overoxidized polypyrrole and gold nanoparticles and capped the transglutaminase layer with bovine serum albumin. As a result, good surface coverage of the gold nanoparticles (~100 nm) was achieved across the overoxidized polypyrrole film. Electrochemical impedance spectroscopy parameters of the gold nanoparticle–overoxidized polypyrrole electrode include time constant, exchange current density, and heterogeneous rate constant. The impedimetric immunosensor exhibited a charge transfer resistance-dependent linear range with a correlation coefficient value of 0.98 and a dynamic linear range of 10⁻⁶–10⁻⁴ mg mL⁻¹. The synergistic effect of the gold nanoparticles on the overoxidized polypyrrole formed the electronic insulator platform as a selective accumulator for the analytes surrounding the nanoelectrode. This biosensor characteristic enabled detection of antitransglutaminase antibodies at very low concentrations without the aid of a secondary label.