This article reported the preparation of an amphoteric natural polymeric membrane-macroporous chitosan (CS)/carboxymethylcellulose (CMC) blend membrane and the utilization of such a membrane on the membrane chromatography for bioseparation. The membranes were prepared by solution blending of CS and CMC solution, and using silica particles as porogen. Both glutaraldehyde and epichlorohydrin were used as crosslinking agent to increase its chemical stability in aqueous solution. Such a natural polymeric membrane can be served as an amphoteric membrane because of the amino group on CS and the carboxymethyl group on CMC, in which the surface charge can be changed with the environmental pH. Ovalbumin (pI = 4.6) and lysozyme (pI = 11) were selected as model proteins. These two proteins adsorption on different CS/CMC blend membranes with different initial protein concentrations at different pH values were investigated in batch systems. The results indicated that the maximum adsorption for lysozyme and ovalbumin was at pH 9.2 and 4.8 respectively, and the adsorption capacity on the membrane both increased with the increase of initial protein concentration. Though the adsorption mechanism of lysozyme and ovalbumin was found not the same, the maximum adsorption capacity of two proteins on the membranes was quite similar (about 250 mg/g). Moreover, the desorption ratio of both proteins was found to be more than 90% that implied CS/CMC blend membrane could separate proteins by adsorption-desorption process. Finally, both lysozyme and ovalbumin were successfully separated from their binary mixture only by adjusting the pH of the feed and the desorption solution.