In this article, we developed novel immobilized Cu2+-hydroxamic acid functionalized poly (methyl methacrylate) grafted cellophane membranes for affinity separation of His-Tag chitinase. First, we grafted the cellophane membranes with poly (methyl methacrylate) (PMMA) and then treated with hydroxylamine hydrochloride to induce hydroxamic acid ions exchange groups, and so we immobilized the copper ions on the hydroxamic acid functionalized PMMA grafted membranes. Fourier transforms infrared analysis and thermal gravimetric analysis provided pieces of evidence of the grafting and the functionalization processes. The membranes show a high affinity toward separating the chitinase enzyme from BSA mixtures. The Cu2+-hydroxamic acid functionalized poly (methyl methacrylate) grafted cellophane membranes show no leakage of Cu2+ ions in the eluting solution during the protein elution process while eluted 88% of the adsorbed chitinase and 80% of the adsorbed BSA.