The goal of this study was to design and construct a biosensor for detecting domoic acid (DA) using bioimmobilization of DA antibodies on the surface of screen‐printed carbon electrodes (SPCEs) enhanced with gold nanoparticles (AuNPs). To accomplish this aim, the SPCE surface was modified by applying AuNPs using electrodeposition, and the optimum modification time was determined by using cyclic voltammetry (CV). Electrochemical impedance spectroscopy (EIS) was performed to thoroughly analyze the electrochemical alterations in SPCEs prior to and during the modification with AuNPs. In addition, we conducted a comprehensive analysis of the structural surface characteristics, topography, and contact angle measurements. The limit of detection (LOD) for the AuNPs‐modified SPCE was determined to be 1.069 ng/mL, whereas the limit of quantification (LOQ) for DA was found to be 3.52 ng/mL using the EIS technique. While many studies concentrate on identifying target molecules through nanoparticle modification, our research surpasses this by offering a comprehensive electrochemical analysis of the modification of AuNPs and a thorough assessment of the changes in the microstructure of the electrode surface. This strategy greatly enhances the progress of biosensor development in the area. In addition, we investigated the harmful effects of DA on human endothelial cells EA.hy926 by subjecting them to different concentrations ranging from 0.001 to 1 ng/mL for a duration of 24 h. This experiment demonstrated that there was a decrease in cell viability that was directly proportional to the concentration of DA.