Novel nanocomposite films containing DNA-silver nanohybrids have been successfully fabricated by combined use of die layer-by-layer self-assembly technique and an in situ electrochemical reduction method widi the DNA-Ag + complex as one of the building blocks. UV-vis absorption spectroscopy was employed to monitor the buildup of die multilayer films, which suggested a progressive deposition widi almost an equal amount of the DNA-Ag+ complex in each cycle. The following electrochemical reduction of silver resulted in the formation of metal nanoparticles in the film, which was evidenced by die evolution of die intense plasmon absorption band originating from silver. Scanning electron microscopy indicated that die particles formed in the multilayer films possessed good monodispersity and stability, thanks to die surrounding polymers. X-ray photoelectron spectroscopy further confirmed die presence of the main components (such as DNA and metallic silver) of the nanocomposite films. In addition, we show that the size of the metal nanoparticles and the optical property of the film could be readily tuned by manipulating the assembly conditions. Furthermore, the feasibility of the as-prepared nanocomposite films functioning as a surfaceenhanced Raman scattering active substrate for sensing purposes was investigated, and die results showed great enhancement of die Raman signal of two probe molecules, Rhodamine 6G and 4-aminothiophenol.