We utilize the extracted water-soluble wool keratin as the building block for the layer-by-layer (LbL) assembly. By adjusting the solution pH, keratin can be used as polycation or polyanion. Experimental results indicate that multilayer films of keratin together with other synthetic polyelectrolytes have been fabricated successfully. We have found that the average thickness of each bilayer of the keratin containing film is evidently larger than that of usual multilayer films previously reported. This feature is helpful to fabricate relatively thick films in less deposition cycles. The use of keratin in LbL assembly is helpful to prepare a biocompatible surface for tissue engineering.