We report on a general lithography method for high-resolution biomolecule patterning with a bilayer resist system.Biomolecules are first immobilized on the surface of a substrate and covered by a release-and-protection interlayerof water-soluble polymer. Patterns can then be obtained by lithography with a spin-coated resist layer in a conventionalway and transferred onto the substrate by reactive ion etching. Afterward, the resist layer is removed by dissolutionin water. To demonstrate a high-resolution patterning, soft UV nanoimprint lithography has been used to producehigh-density dot arrays of poly-(l-lysine) molecules on a glass substrate. Both fluorescence images and cell proliferationbehaviors on such a patterned substrate have shown evidence of improved stability of biomolecule immobilizationcomparing to that obtained by microcontact printing techniques.