In this study, a strategy for the construction of nanocomposite multilayer films was developed. Using alternate processes of electrodeposition and self-assembly, ordered multilayer films of Prussian blue and multiwalled-carbon nanotube/polyethylenimine/Au (MWNTs-PEI-Au) nanocomposites were successfully fabricated onto the surface of Au electrode. The feasibility of this alternate manner was confirmed by UV-Vis spectrometry, scanning electron microscopy and electrochemical measurements. In addition, the electrochemical properties of the constructed nanocomposite film were investigated. Here, the fabricated sandwich-like multilayer films demonstrated rather high stability and sensitivity to hydrogen peroxide due to the multilayer film structure and the catalytic ability of stable PB layer. Under optimal experimental conditions, the fabricated sensor responded to hydrogen peroxide in a linear range from 0.5 mu M to 3.43 mM, with a low detection limit of 80 nM. The sensitivity of the sensor was found to be 2.27 mA/mMcm(2). The present work demonstrates an alternate manner of electrodeposition and self-assembly that shows promising potential for developing new biosensors. (C) 2011 The Electrochemical Society. [DOI: 10.1149/2.037202jes] All rights reserved.