Although molecularly imprinted materials using small organic molecules as templates have been well established, development of such materials for protein separation is still rather challenging. We therefore describe herein a two-step surface imprinting method established with a hydrophilic macroporous cellulose membrane with relatively large specific surface area. In the first step, tailor-made multi-functional polymer chains were grafted on the cellulose membrane using photo-initiated graft copolymerization, enabled by a surface-immobilized photo-initiator. This scaffold allowed the preorganization of the template protein lysozyme (Lys) on the surface of the membrane pores. Notably, the scaffold-grafted membrane showed already a significant adsorption selectivity versus the very similar protein cytochrome C (CyC). In the second step, surface-initiated cross-linking copolymerization, enabled by a photo-initiator immobilized in the scaffold layer, resulted in a protein-imprinted cellulose membrane. Imprinting efficiency was further improved by optimization of monomer concentrations in the second step. Protein selectivity of the best imprinted cellulose membrane for Lys over CyC reached a very remarkable value of about 45, measured with 1 : 1 mixtures of the two proteins. We envision that this property of a protein-imprinted cellulose membrane, which is based on the tailored binding selectivity achieved using the two-step functionalization method, could be largely beneficial for separation and purification of target proteins from complex mixtures.