Protein thin films containing avidin were successfully prepared by a layer-by-layer deposition of avidin and concanavalin A (Con A) on the surface of a quartz slide, through a strong bioaffinity between Con A and sugar chains attached to the surface of avidin molecule. The UV-visible absorption spectroscopy and quartz-crystal microbalance studies revealed that avidin forms nearly a monomolecular layer in each layer of the multilayer films. Avidin molecules built into the multilayer films retained their binding activity to biotin and analogue 2-((4'-hydroxyphenyl)azo)benzoic acid. The avidin-Con A films bound biotin-tagged dye (fluorescein) tightly. The avidin-Con A multilayer films are stable in buffer solution or in low concentration of glucose and mannose solutions (10 mM).