Films consisting of various numbers of alternating molecular layers of human serum albumin (HSA) and heparin were prepared on surfaces of polyethylene, polyurethane, cellulose acetate, germanium, hydrophilic and hydrophobic silica, and mica by alternating the HSA and heparin adsorption from solutions at pH 3.9. The successive adsorption of the molecular layers was observed in situ by IR multiple internal reflection spectroscopy and total internal reflection fluorescence. The first HSA monolayer was irreversibly adsorbed on the substrates mainly due to the hydrophobic interaction with the surface, and the following heparin and HSA layers were successively bound due to ionic interaction between HSA and heparin. Films stable in physiological solutions were obtained by the covalent crosslinking of the final HSA-heparin assembly.