Protein-containing polyelectrolyte multilayer films of poly(styrenesulfonate) and poly(allylamine hydrochloride), fabricated by the sequential adsorption of polyelectrolyte and anti-immunoglobulin G (anti-IgG) on solid substrates, have been characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), and Fourier transform infrared reflection-absorption spectroscopy (FTIR-RAS). Visualization of the film structure on the nanometer scale, by AFM and SEM, showed that either layered or disordered films were formed depending on the number of polyelectrolyte layers separating each protein layer. For films where each anti-IgG layer was separated by one polyelectrolyte layer, an open, disordered film structure was observed and significant protein aggregation occurred. In contrast, for films in which the anti-IgG layers were separated by five polyelectrolyte layers, a layered structure with uniform protein layers was formed. Film thicknesses determined by SEM measurements were consistent with those calculated from quartz crystal microbalance measurements. FTIR-RAS confirmed the presence of anti-IgG in the multilayer films, with the amide I and II bands due to anti-IgG clearly visible in the spectra, and provided direct evidence that anti-IgG was not denatured. Both types of films fabricated are interesting for biosensing applications : the first provides ordered, functional protein layers within a polyelectrolyte matrix for sensing investigations, and the second serves as a useful functional film for applications where an increased binding capacity of the film is sought.