Histamine (HA) is a biogenic amine that can accumulate to high concentration levels in food as a result of microbial activity and can cause toxic effects in consumers. In this work, a portable electrochemical immunosensor capable of detecting HA with high sensitivity and selectivity was developed. Prussian blue-chitosan-gold nanoparticle (PB-CS-AuNP) nanocomposite films with excellent biocompatibility were synthesized and characterized by scanning electron microscopy and energy dispersive X-ray analysis. The PB-CS-AuNP were coated onto a screen-printed electrode by one-step electrodeposition and used to conjugate the HA ovalbumin conjugate (HA-Ag). HA was determined by a competition between the coating HA-Ag and the HRP labeled HA antibody (HRP-HA-Ab). After careful optimization of assay conditions and Box-Behnken analysis, the developed immunosensor showed a linear range from 0.01 to 100μg/mL for HA in fish samples. The average recoveries from spiked samples ranged from 97.25% to 105%. The biosensor also showed good specificity, reproducibility, and stability, indicating its potential application in monitoring HA in a simple and low cost manner. •A portable immunosensor for sensitive detection of histamine was developed.•Prussian blue-chitosan-gold nanoparticles were used as signal amplification materials.•The immunosensor showed a detection limit of 1.25ng/mL for histamine in fish samples.