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Wheat gliadin is thought to be the main cause of wheat allergy leading to celiac disease. In this study, we reported an electrochemical immunosensor based on AuNPs/Zn/Ni-ZIF-8-800@graphene for simple, efficient, and sensitive detection of gliadin. A monoclonal antibody against gliadin were prepared by hybridoma technique. Zn/Ni-ZIF-8-800 with high stability was prepared by using zinc–nickel bimetallic organic framework MOFs, and graphene and gold nanoparticles with large surface area and excellent conductivity were introduced. Chitosan was used as a binder to fixed the composite on the surface of glassy carbon electrode. The fabricated materials and the corresponding sensors were comprehensively characterized by X-ray diffractometer, scanning electron microscope, transmission electron microscopy, Fourier transform infrared spectroscopy, Brunauer–Emmett–Teller, and cyclic voltammetry analyses. Under the optimal conditions, the linear range of the electrochemical immunosensor was 0.1–100 μg mL−1, and the detection limit was 0.950 μg mL−1. In addition, the fabricated sensor exhibited high selectivity and long-term stability. The recovery test showed that the prepared sensor was suitable for the detection of gliadin in real food and feed, exhibiting great promising of the developed sensor in practical application. [ABSTRACT FROM AUTHOR]