Adsorption crossflow filtration is a technique that combines the advantages of membrane filtration and chromatography, both widely used to separate and purify the protein mixture in bioseparation processes. Ion exchange adsorption coupled with crossflow filtration for enhancing bovine serum albumin (BSA), ovalbumin (OV) and lysozyme (LY) separation were conducted experimentally in this study. The DEAE Sepharose, diethylaminoethyl weak anion exchange gel particle, was used to examine adsorption behavior under different solution conditions. Protein recovery, purity and transmission during the adsorption crossflow filtration process were analyzed based on the condition of an optimal pH value and ionic strength obtained in the adsorption test. The experimental results reveal that the hydrophilicity difference between BSA and OV leads to different adsorption behaviors at optimal separation conditions of phosphate buffer of pH 9.0 and the ionic concentration 20 mM. As ionic strength in the solution increases, compression of an electrical double layer and electrostatic shielding result in decreasing BSA and OV adsorption capacity. Adsorption operation consistently shows a lower LY capacity since LY and the gel particle have the same charged polarity at the optimal value of the pH 9.0. A two-stage adsorption crossflow filtration process is proposed and examined for protein purification under optimal conditions obtained in the purification of a binary protein mixture solution. Comparison of the experimental results obtained in the two-stage adsorption crossflow filtration in our previous work by crossflow ultrafiltration indicated that adsorption crossflow filtration provides higher efficiency and recovery for protein purification.