To ensure food quality and prevent histamine (HA) toxicity, a rapid and direct method of detecting HA is required. In this work, we prepared a monoclonal antibody (mAb) against HA using a hapten produced by the introduction of a phenyl-containing linker. The novel mAb exhibited high sensitivity against HA as determined by ELISA, with a half-maximal inhibitory concentration of 21.51 ng/mL. A gold nanoparticle-based immunosensor was fabricated for rapid detection of HA in fish samples. After optimizing the immunosensor, a visual limit of detection (LOD) and a calculated LOD were 0.25 mg/kg and 10.48 μg/kg for HA, respectively. Recovery rates from the spiked fish samples ranged from 87.33% to 104.67% with the coefficient of variation below 10.82%. Concurrently, the whole process in testing real sample was completed within 15 min, and all results were well confirmed and comparable by liquid chromatography-mass spectrometry and the commercial test strip. These data revealed that the proposed immunosensor could be used as a monitoring tool for the rapid and direct detection of HA in fish samples.