A new series of chemically well-defined brush glycopolymers consisting of a polyoxyethylene backbone and bristles bearing glycosyl and methyl end groups was synthesized with various compositions. The glycopolymers were thermally stable up 200 °C and were soluble in a variety of common solvents. The brush polymer films formed multibilayer structures, the layers of which were stacked along the direction normal to the film plane so as to display a glycosyl group-rich surface or a methyl group-rich surface or their mixture, depending on the bristle end group composition. The multibilayer structures were stabilized by the self-assembly of the bristles via lateral packing. The glycosyl-rich surface played a critical role in enhancing the surface hydrophilicity and water sorption to a certain level; thus, the glycopolymer films easily formed a hydration layer to a certain depth on the film surface. The hydrophilic surfaces and hydration layer efficiently prevented protein adsorption onto the brush glycopolymers and suppressed bacterial adherence while promoting mammalian cell adhesion and displaying excellent biocompatibility in an in vivo mouse study.