In this article, highly rough and stable surface enhanced Raman scattering (SERS)-active substrates had been fabricated by a facile layer-by-layer technique. Unique lambda-DNA networks and CTAB capped silver nanoparticles (AgNP) were alternatively self-assembled on the charged mica surface until a desirable number of bilayers were reached. The as-prepared hybrid architectures were characterized by UV-vis spectroscopy, tapping mode atomic force microscopy (AFM) and confocal Raman microscopy, respectively. Linear increases of the maximum absorbance of DNA band with the number of bilayers present a common LBL assembly feature. The red-shift of surface plasmon of silver nanoparticles within the hybrid films was mainly due to the aggregation effect. With the increase of number of bilayers, the surface coverage of nanoparticles on the substrate became larger, as well as the rising of total amount of nanoparticles and the surface roughness of hybrid films. These rough metallic hybrid architectures could be utilized as SERS-active substrates. A significant enhanced Raman scattering effect of the adsorbed analytes, e.g., methylene blue (MB), on these hybrid films was easily exploited by the confocal Raman microscopy. The enhancement factor depended on the surface coverage of nanoparticles and number of bilayers of lambda-DNA/AgNP.