A new and effective strategy for constructing a mediator-type amperometric immunosensor based on the unique characteristics of DNA-PDDA polyion complex membrane and gold nanoparticles was described. A poly(toluidine blue O) (PTOB) film deposited on a DNA-PDDA polyion complex membrane surface, exhibited excellent electrochemical redox property. Stable and well-defined redox peaks for the PTOB redox couple were obtained in pH 6.5 phosphate buffer solution (PBS). The introduction of DNA-PDDA polyion complex film not only enhanced the electrode surface area for construction of efficient biosensors, but also acted as a charge carrier to facilitate the electron transfer. Moreover, as a host matrix, the DNA-PDDA polyion complex could firmly immobilise the PTOB membrane on the surface of electrode by strong interaction. With carcinoembryonic antigen (CEA) as a model antigen, the presence of gold nanoparticles provided a congenial microenvironment for adsorbing biomolecules and decreased the electron transfer impedance. The fabrication process of the immunosensor was characterized by atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immunosensor displayed a high-sensitivity for the detection of CEA and had good correlation for the detection of CEA in the range of 0.5-120.0 ng/ml with a detection limit of 0.3 ng/ml (estimated at a signal-to-noise ratio of 3). The proposed immunosensor showed a broader linear range, good reproducibility, and storage stability. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.