Multilayer thin films composed of a positively charged protein and cationic polymer layers can be prepared on a solid surface against their electrostatic force of repulsion, by depositing avidin and biotin-labeled polymers [poly(ethyleneimine) (PEI), poly(allylamine) (PAA), or poly(amidoamine) (PAMAM) dendrimer] alternately and repeatedly. In contrast, the thin film does not form when unmodified polymers are used, confirming that the strong binding between avidin and biotin is responsible for the formation of the multilayer films. The loading of avidin in each layer of the multilayer films depends significantly on the molecular geometry of the polymers. The avidin/PAMAM dendrimer films are composed of a monomolecular layer of avidin whereas the avidin multilayers are formed in each layer of the PAA- and PEI-based thin films. The results are rationalized by the different molecular structures of the polymers; PAMAM dendrimer assumes a globular shape, as compared with the linear and highly branched polymer chains of PAA and PEI, respectively.