Immobilized Cu(2+) ions affinity cellophane-poly(glycidyl methacrylate) (PGMA)-grafted membranes have been prepared through three steps. The first step was introducing of epoxy groups to its chemical Structure through grafting process with PGMA. Factors affecting the grafting process have been studied and grafting percentage (GP) up to 233%, has been obtained. The second step was converting the introduced epoxy groups to sulfonic ones. It was found that maximum amount of sulfonic groups (2.7 mmol/g) was obtained with minimum GP (46.08%). The third and last step was the immobilization of Cu(2+) ions into sulfonated grafted membranes obtained from the previous step. Maximum amount of immobilized Cu(2+) ions was found to be 60.9 ppm per grain of polymer. The verification of the grafting and sulfonation steps has been performed through characterization of the obtained membrane using FTIR, TGA, and EDAX analysis. Finally, Cu(2+)-immobilized membranes have been evaluated in separation of P-galactosidase (beta-Gal) enzyme from its mixture with bovine serum albumin (BSA) in different pH medium. Maximum protein adsorption, for both proteins, has been obtained at pH range 4-4.5; as 90 and 45%, for beta-Gal and BSA, respectively. The results showed high affinity toward beta-Gal separation although BSA concentration (0.5%) is 20-folds of beta-Gal (0.025%,). (C) 2008 Wiley, Periodicals, Inc. J Appl Polym Sci 111: 2647-2656, 2009