Histamine can be formed by enzymatic decarbonylation of histidine, which is an important indicator of seafood quality. A rapid and sensitive assay method is necessary for histamine monitoring. A fluorescence resonance energy transfer (FRET) assay system based on a carbon dot (CD)-modified nanoporous alumina membrane and Fe3O4@Au magnet nanocomposites has been developed for histamine detection in mackerel fish. CDs immobilized on nanoporous alumina membranes were used as donors, which provided a fluorescence sensing substrate for histamine detection. Fe3O4@Au magnet nanocomposites can not only act as acceptors, but also concentrate histamine from fish samples to increase detection sensitivity. Histamine was detected by the fluorescence signal changes of CDs capturing histamine by an immune reaction. The fluorescence signals of CDs were quenched by Fe3O4@Au magnet nanocomposites via the FRET mechanism. With an increase of histamine, the fluorescence intensity decreased. By recording fluorescence spectra and calculating intensity change, histamine concentration can be determined with a limit of detection (LOD) of 70 pM. This assay system can be successfully applied for histamine determination in mackerel fish to monitor the fish spoilage process in different storage conditions. It shows the potential applications of CDs-modified nanoporous alumina membranes and Fe3O4@Au magnet nanocomposites-based biosensors in the food safety area.