Bimetallic (Poly(ethylenimine) (PEI)-Ag/Au) multilayer film was in situ simultaneously fabricated by alternating immersions of a substrate in PEI-Ag+ and AuCl4- solutions followed by chemical reduction with NaBH4 solution. In the process, the AuCl4- ions not only play an important role of a reaction reagent, but also served as an assembly reagent. Au. Ag nanoparticles (NPs) were observed with a spherical morphology and well-dispersed in the composite multilayer film, and the size of Au NPs in the bimetallic {PEI-Ag/Au) multilayer film was smaller than that of the single Au NPs formed in {PEI/Au) multilayer films. It was also very interesting to observe that this bimetallic (PEI-Ag/Au) multilayer film exhibited more efficient electrocatalytic activity for the oxidation of ascorbic acid than the multilayer film containing only single Au or Ag NPs. These results indicated that this bimetallic composite multilayer film may be potentially applied in electrochemical biosensors. (C) 2011 Elsevier B.V. All rights reserved.