Immobilized Cu2+ ions affinity cellophane membranes for affinity separation of His-tag Chitinase were prepared. Cellophane membranes were first grafted with polymethacrylic acid, and the impacts of various grafting conditions on grafting process parameters were evaluated. Fourier transform infrared analysis and thermal gravimetric analysis provided evidences of grafting occurrence. The grafted membranes were then immobilized with copper ions as the last step before being tested in separation of chitinase enzyme from an equal protein mixture with bovine serum albumin (BSA). The membranes show high affinity toward the separation of chitinase enzyme, which adsorbed 80% of chitinase and only 28% of BSA from the 0.25% protein mixture solution. At lower concentrations, 0.025%-0.05%, the membranes show complete selectivity toward chitinase. No leakage of Cu2+ ions was detected in the elution solution, whereas 95% of chitinase and 90% of BSA were eluted. (C) 2011 Wiley Periodicals, Inc. Adv Polym Techn 30: 191-202, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/adv.20216