A label-free electrochemical impedimetric immunosensor has been developed for the rapid and sensitive detection of zearalenone (ZEN) by immobilizing monoclonal anti-zearalenone antibodies (ZEN-MAb) onto the palladium nanoparticle embedded functionalized carbon nanotube-chitosan (SPCE/CS-CNT-Pd) modified electrode. This CS-CNT-Pd nanocomposite consists of finite Pd nanoparticles implanted onto the covalently functionalized CNTs by the chemical reduction method. The structure of the nanocomposite was studied by using X-ray diffraction (XRD), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDX). The major advantages of the coexistence of Pd nanoparticles with CNTs are greatly enhanced conductive, catalytical properties of transducer surface, and better immobilization of bio-recognition antibody. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to verify the stepwise assemblage of the developed immunosensor. The immobilization of antibodies and the binding of zearalenone to the modified electrode increased the electron-transfer resistance (Rct), which was directly measured with EIS using [Fe(CN)6]3/4- as a redox probe. The developed immunosensor was quite sensitive and simple compared with various conventional methods. The immunosensor performance was tested under the optimized conditions, towards the detection of ZEN, from 0.25 to 16 ng mL−1 with a detection limit of 0.25 ng mL−1. The new biocompatible CS-CNT-Pd based immunosensor was successfully applied to assay ZEN in corn samples, and the good recovery values were gained, this holds the considerable promise for the sensor performance in the food industries. © 2022