In this paper, we report a new concept to construct a label-free electrochemical inhibition-based immunosensor for the detection of the mycotoxin deoxynivalenol (DON) in cereal samples. The electrochemical impedance spectroscopy of tris(bipyridine) ruthenium (II) chloride was used as a marker enhanced with gold nanoparticles-dotted 4-nitrophenylazo functionalized graphene (AuNp/G/PhNO2) nanocatalyst mediated in Nafion on a glassy carbon electrode. Under the optimized conditions, the formation of immunocomplexes inhibited electron flow and increased the charge transfer resistance of the sensing interface linearly. The change in impedance was proportional to DON concentrations in the range of 6-30 ng/mL with a sensitivity and detection limit of 32.14 ΩL/ng and 0.3 µg/mL, respectively, which compares favorably with the ELISA result. The proposed sensor had a stability of 80.3%, good precision and selectivity in DON standard solution containing different interfering agents, indicating promising application prospect for this strategy in designing impedimetric, electrochemiluminescent, voltammetric or amperometric sensors.