An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe 3 O 4 /polyaniline/Nafion (Fe 3 O 4 /PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab 2 ) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe 3 O 4 /PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe 3 O 4 /polyaniline (Fe 3 O 4 /PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8pM and 2nM with the detection limit of 4pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants.