Chromatography is a widely used technique in protein separation, and the adsorbents are essential to separate target proteins from raw mixtures. In this study, porous cellulose beads were prepared with a direct dissolution of microcrystalline cellulose (MCC) using an ionic liquid solvent 1-butyl-3-methylimidazolium chloride. Pore expanding agents (cassava starch and cyclohexane) were added in MCC solutions to change the pore structure of the cellulose beads. The results showed that the mean pore size of cellulose beads increased after the application of pore expanding agents, whereas the wet density and the specific surface area were decreased. Residence time distribution studies indicated that the beads prepared with the addition of cyclohexane had the best performance for a series of molecules with different molecular weights. The cellulose beads were coupled with diethylaminoethyl and the adsorption properties with bovine serum albumin as a model protein showed that the beads prepared with cyclohexane had the best protein adsorption capability. The chromatographic results demonstrated that ionic liquids are effective solvents for cellulose dissolution and pore expanding agents can be used to enhance the pore structure of cellulose beads. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40060.