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 interlayer of water-soluble polymer. Patterns can then be obtained by lithography with a spin-coated resist layer in a conventional way and transferred onto the substrate by reactive ion etching. Afterward, the resist layer is removed by dissolution in water. To demonstrate a high-resolution patterning, soft UV nanoimprint lithography has been used to produce high-density dot arrays of poly-(L-lysine) molecules on a glass substrate. Both fluorescence images and cell proliferation behaviors on such a patterned substrate have shown evidence of improved stability of biomolecule immobilization comparing to that obtained by microcontact printing techniques.