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Abstract: A novel label-free electrochemical immunosensor for sensitive detection of kanamycin based on water-soluble graphene sheet (WGS)/prussian blue-chitosan (PB-CTS)/nanoporous gold (NPG) composited film has been reported. PB was selected as an electron transfer mediator, and was modified onto the electrode together with WGS through electrostatic adsorption. Then NPG was immobilized onto the as-prepared film for biomolecules anchoring. The electroactivity of PB was greatly enhanced in the presence of WGS and NPG. It could mainly be ascribed to the fact that the good conductivity of WGS and NPG promoted electron transfer and enhanced the sensitivity. kanamycin antibody, as a model, was immobilized onto the composite film for the detection of kanamycin. Under optimum conditions, the amperometric signal of PB decreased linearly with kanamycin concentration (0.02–14ngmL−1), a linear calibration plot (y =1.3817+4.7544x, r =0.9993), resulting in a low limit of detection (6.31pgmL−1). The novel immunosensor for the detection of kanamycin in real sample with satisfactory results has been proved. In addition, this method would be easily adapted for the detection of other residual antibiotics in animal derived foods. [Copyright &y& Elsevier]