A label-free amperometric immunosenor for determination of anthracene was developed. Prussian blue was electrodeposited on the glassy carbon electrode and then modified with nanometer-size gold film to protect Prussian blue layer and immobilized anthracene polyclonal antibodies. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry. The performances of the amperometric immunosensor were studied in detail. At the optimal experimental condition, the current response of the immunosensor was proportional to the concentration of anthracene in the range of 1.8~90 ng•mL-1 with a detection limit of 0.82 ng•mL-1 (S/N=3). The studied immunosensor exhibited low detection limit, long-term stability and easy regenerated. This mothod was applied to the anthracene detection in river water samples with satisfied results.