Multilayer thin films of ∼ 7 nm diameter gold nanoparticles (GNPs) linked with horse heart myoglobin (Mb) are fabricated, for the first time, by layer-by-layer (LbL) assembly on glass slides, and silicon and plastic substrates. The GNP/Mb nanocomposite films show sharp surface plasmon resonance (SPR) absorption bands that are used to follow the LbL growth of the film and to determine the kinetics of GNP adsorption on the Mb-modified surface. The GNP/Mb nanocomposite films are characterized using atomic force microscopy, transmission electron microscopy, polarized UV-vis spectroscopy, and spectroscopic ellipsometry. The GNPs in the multilayer films are spatially separated from one another, and interparticle interactions remain in the film, making it optically anisotropic. The GNP/Mb nanocomposite films are stable in air at temperatures up to 100 °C, and can withstand successive immersions in strongly acidic and basic solutions. The SPR absorption band of the GNP/Mb nanocomposite film in air exhibits a red-shift in the wavelength maximum and an increase in the maximum absorbance relative to that in water. This result, which is in contrast to that observed with a GNP monolayer on an aminosilane-functionalized substrate, suggests the shrinkage in air and swelling in water of Mb molecules embedded in the nanocomposite film.