© 2020 Wiley-VCH GmbHAlthough commercial screen-printed electrodes (SPEs) are used extensively for biosensor purposes nowadays, detailed studies on characterization are still limited. In this study, the surface of the gold-based screen-printed electrode (SPGE) was carefully modified with self-assembly-monolayer through an optimized immobilization procedure. The key physical and chemical properties with regeneration capacity of the developed biosensors were assessed by various characterization techniques. Then SPGE was used to determine its sensitivity, limit of detection (LOD) and limit of quantification (LOQ) for a toxin substance of domoic acid in seafood that has become more common and rising concern of marine wildlife and seawater pollution. LOD in phosphate buffered saline (PBS) and cell culture media were obtained as 2.93 ng mL−1 and 4.28 ng mL−1, respectively. The reduced sensitivity for antibody-based biosensors in the cell culture medium was probably due to interaction of nonspecific compounds with DA in the culture medium compared to the much less complex environment of PBS. In addition, the regeneration capacity has been found very limited due to inherent heterogeneity and low robustness. This study can be used for the main challenges with the design requirements of commercial SPE-based biosensors to provide a detailed perspective for further toxicity studies.