In this study, we describe the first demonstration of well-ordered multilayer thin films of gold nanoparticles through a layer-bylayer (LbL) technique assisted by a polypeptide template. Anionic poly(L-glutamic acid) (PLGA)-modified gold nanoparticles were prepared as building units for LbL self-assembly. Two types of synthetic polymers, poly(L-lysine) (PLL) and poly(allylamine), were used as polycations that carry the same amino groups at the side chain, although the former is a polypeptide and the latter is a vinyl polymer. The deposition process and the resultant multilayer films were characterized by means of absorption, circular dichroism, Fourier transform infrared and atomic force microscopy measurements. Experimental results revealed that LbL multilayer properties, such as thickness, conformation of the peptide component, homogeneity and periodicity of nanoparticle layers, were significantly affected by the kind of polycations present. In particular, the combination of anionic PLGA-coated gold nanoparticles and cationic PLL induced b-sheet formation, which enabled the fabrication of a homogeneously well-packed gold nanoparticle multilayer without aggregation.