A glycocalyx-mimetic film was created by the assembly of biotin chain-terminated glycopolymers onto a polymeric lipid membrane electrostatically coupled to a polyelectrolyte multilayer. Varying molar compositions (10, 25, and 50 mol %) of acrylate-derivatized biotin-phosphoethanolamine/phosphorylcholine lipid mixtures were prepared as unilamellar vesicles, fused onto the alkyl chains of an amphiphilic terpolymer, and photopolymerized in situ as a planar assembly. Polarized external reflection infrared spectroscopy confirmed the presence of streptavidin. Notably, IR spectroscopy revealed an increase in the conformational and orientational disorder of the lipid hydrocarbon chains with increasing mole fraction of biotinylated lipid. Correlative images obtained by confocal fluorescence microscopy demonstrated that biotinylated lipids cluster at high surface density. Despite the presence of biotin microdomains, the size and hydrophilic characteristics of coupled glycopolymer chains produced a uniform carbohydrate surface coating.