A sensitive electrochemical immunosensor was prepared for rapid detection of ASA based on arsanilic acid (ASA) monoclonal antibody with high affinity. In the preparation of nanomaterials, polyethyleneimine (PEI) improved the stability of the solution and acted as a reducing agent to generate reduced graphene oxide (rGO) with relatively strong conductivity, thereby promoting the transfer of electrons. The dual conductivity of rGO and silver nanoparticles (AgNPs) improved the sensitivity of the sensor. The synthesis of nanomaterials were confirmed by UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy and scanning electron microscopy. In the optimal experiment conditions, the sensor could achieve the detection range of 0.50–500 ng mL⁻¹ and the limit of detection (LOD) of 0.38 ng mL⁻¹ (S/N = 3). Moreover, the sensor exhibited excellent specificity and acceptable stability, suggesting that the proposed sensor possessed a good potential in ASA detection. Thus, the as-prepared biosensor may be a potential way for detecting other antibiotics in meat and animal-derived foods.