Gold nanoparticles (Au-NPs) functionalized Poly (3,4-ethylenedioxythiophene) (PEDOT) bioelectrodes were fabricated layer by layer deposition on ITO electrode for detection of aflatoxin B. The monoclonal anti-aflatoxin antibodies (Anti-AFB) were immobilized over the surface of PEDOT/Au-NPs/ITO using EDC/NHS coupling. The surface morphology and characteristics of the modified electrodes were investigated by scanning electron microscope and contact angle measurements. The electrochemical analysis of the fabricated immunoelectrode and the immobilization of the antibodies have been evaluated and confirmed by performing Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Fourier Transform Infrared Spectroscopy (FTIR). Decrease in the value of electron transfer resistance (R) and increase in the peak current values after incorporation of Au-NPs signifies the enhanced properties of PEDOT embedded Au-NPs. The heterogeneous rate constant (k) and transfer coefficient (α) have been determined by using Laviron's method. The fabricated immunosensor exhibits high sensitive amperometric response of 3.72μAngmL towards AFB concentration in a linear range of 1-25ngmL with detection limit (LOD) of 0.0045ngmLand limit of quantification (LOQ) of 0.0156ngmL. The fabricated immunoelectrode shows a reproducibility of 96.13% and 94.5% towards real maize sample spiked with AFB of concentration 30ngmLand 50ngmL, respectively.