Mixed matrix membranes (MMMs), which incorporate adsorptive particles during membrane casting, can be prepared simply and have performances that are competitive with other membrane chromatography materials. The application of MMM chromatography for fractionation of beta-Lactoglobulin from bovine whey is described in this article. MMM chromatography was prepared using ethylene vinyl alcohol polymer and lewatit anion exchange resin to form a flat sheet membrane. The membrane was characterized in terms of structure and its static and dynamic binding capacities were measured. The optimum binding for beta-Lactoglobulin was found to be at pH 6.0 using 20 mM sodium phosphate buffer. The MMM had a static binding capacity of 120 mg/g membrane (36 mg/mL membrane) and 90 mg/g membrane (27 mg/mL membrane) for beta-Lactoglobulin and alpha-Lactalbumin, respectively. In batch fractionation of whey, the MMM showed selective binding towards beta-Lactoglobulin compared to other proteins. The dynamic binding capacity of beta-Lactoglobulin in whey solution was about 80 mg/g membrane (24 mg beta-Lac/mL of MMM), which is promising for whey fractionation using this technology. This is the first reported application of MMM chromatography to a dairy feed stream.