Novel fibronectin (FN)-based ultrathin films were prepared by layer-by-layer (LbL) assembly. Among the various combinations of extracellular matrix (ECM) proteins and glycosaminoglycans (GAGs) such as FN, gelatin (G), alpha-elastin (E) and heparin (Hep), FN/Hep, FN/G and FN/E nanofilms were successfully fabricated in phosphate buffer solutions (pH 7.4). The film thickness of the nanofilms, in which each component can interact with each other by FN-specific interactions, was larger than that of other LbL films (E/Hep, G/E and G/Hep) prepared by electrostatic interactions. The FN/G film was rapidly decomposed by treatment with elastase, thus demonstrating, the enzymatic biodegradability of the nanofilm. We prepared the FN/heparinoid multilayers composed of FN and dextran sulfate (Dex), and its thickness was much larger than that of the FN/alpha-poly(L-lysine hydrochloride) (PLL) film prepared by LbL assembly using common electrostatic interactions. Furthermore, the FN/G and FN/Dex nanofilms prepared by FN-specific interaction were more stable in Eagle's MEM with 10% fetal bovine serum (FBS) than the electrostatic assembling films, FN/PLL and PLL/Dex. FN-based multilayers composed of FN and ECM components can be useful as artificial ECM films for tissue engineering and other biomedical applications.