In this work, a sensitive electrochemical immunosensor has been reported for the determination of norfloxacin in animal-derived foods. The poly (amidoamine) dendrimer encapsulated gold nanoparticles (PAMAM-Au) played dual roles in the proposed sensing platform which not only accelerated the electron transfer process of sensing, but also increased the efficiency of the immobilized antibody. The HRP-labeled antigen, as the signal labels in the immunosensor, was introduced to catalyze the following reaction of the substrate hydroquinone with the aid of H₂O₂ in the competitive reaction. On the basis of the signal amplification of PAMAM-Au, the signal intensity was linearly related to the concentration of norfloxacin in the range of 1 μg·L⁻¹–10 mg·L⁻¹. All the results showed that the proposed strategy with low LOD (0.3837 μg·L⁻¹) and favorable recovery (91.6–106.1%) in the practical sample, and it could provide a suitable protocol for norfloxacin detection in animal-derived foods with high sensitivity, good accuracy, and stability.