When consumed in excessive amounts, histamine (HIS), a naturally occurring component in seafood, is known to produce an unpleasant inflammatory reaction. Hence, proper histamine measurement and detection in seafood are of utmost importance. Herein, a histamine electrochemical biosensor based on a molecularly imprinted polymer (MIP) was fabricated using a gold (Au) inverse opal (IO) electrode. Prior to the MIP synthesis, the scaffold of Au IO was modified by electropolymerizing 3,4-ethylenedioxythiophene (EDOT), providing a substrate with improved electrical characteristics. The fabrication of the MIP film was achieved by electropolymerizing aniline in circumstances that preserved the chemical structure of HIS. The electron transport properties of a standard redox probe [Fe(CN) 6 ] 3−/4− were evaluated by making use of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We describe a synergistic strategy for constructing a hierarchical recognition material using an electrochemical approach that successfully integrates three independent approaches, including semicovalent, molecular imprinting, and inverse opal structure, to enhance selectivity and sensitivity. The fabricated HIS electrochemical biosensor exhibited a linear response from 50 nM to 500 μM, a detection limit of 1.07 nM, and was selective against different analytes as well. Moreover, the fabricated HIS electrochemical biosensor outperforms its contemporary counterparts as it also exhibited low error, high accuracy, specificity, stability, and good relative standard deviation % (RSD %).