A novel fluorescent biosensor was developed for the quantitative detection of histamine (Hist), cadaverine (Cad), and putrescine (Put) in seafood. This biosensor is based on the composite material of N,S co-doped carbon dots and manganese dioxide nanoflowers (N,S-CDs@MnO2) and diamine oxidase (DAO). The successful synthesis of N,S-CDs@MnO2 was confirmed through characterization of the synthetic material, and quantitative detection of target biogenic amines was performed by optimizing the detection conditions of the N,S-CDs@MnO2-DAO sensor. The results showed that the linear detection ranges of the fluorescence biosensor for Hist, Cad, and Put were all 0~200 μmol•L-1, and the detection limits were 0.59, 0.65, and 0.85 μmol•L-1, respectively, indicating good method sensitivity. No significant response was observed to amino acids, inorganic salt ions, or reducing substances with potential interference, suggesting good method selectivity. For actual seafood samples, the spiking recovery rate was between 93.17% and 105.34%, and the relative standard deviation was less than 5.83%. For fish, shrimp, and shellfish samples, relative deviation between the detection results of the sensor and HPLC was less than 10%, indicating good accuracy of the method. Therefore, the developed N,S-CDs@MnO2-DAO fluorescent biosensor had good sensitivity, selectivity, and accuracy for the detection of Hist, Cad, and Put. It is an effective tool for the safety and quality control of seafood.