A novel An nanoparticles (Au-NPs)-based protocol for DNA hybridization detection based on assembly of alternating DNA and poly (dimethyldiallylammonium chloride) (PDDA) multilayer films by layer-by-layer (LBL) electrostatic adsorption has been studied. Electrochemical impedance spectroscopy (EIS) and UV-vis absorbance measurements were used to study the film assembly. All the results indicate that the uniform multilayer can be obtained on the polypyrrole (PPy) coated electrode surface and the hybridization reaction can be amplified by the layer-by-layer progress. The hybridization was detected by the reductive signal of Au-NPs and nonspecific adsorption was greatly. eliminated by an unrelated DNA sequence to the tat-get DNA. Under optimum conditions, a significant sensitivity enhancement had been obtained, and the detection limit was down to 3.20 x 10(-14) M when 6 layers assembled. The DNA biosensor has good stability and reproducibility. (C) 2007 Elsevier Ltd. All rights reserved.