Rhodamine B (RB) post-grafted onto beaded cellulose by a curing method is used as a biomimetic ligand in dye affinity chromatography. The grafted materials obtained are qualitatively characterized by scanning electron microscope and fourier transformed infrared spectroscopy. An amount of 76.79 mmol RB/g dyed cellulose is determined by elemental analysis. The RB affinity interaction with the trypsin, alpha-chymotrypsin, and BSA (bovine serum albumin) is analyzed using different mobile phase composition. The results show a selective separation of a mixture of BSA and trypsin into two single peaks by step elution with 1.75 M, 0.5 M, and 0 M ammonium sulphate in the eluent buffer. A good reproducibility of the retention time is obtained for these proteins in the mixture with typical values of 8.0 +/- 0.2 min for BSA and 20.0 +/- 0.2 min for trypsin, showing a possible application in the purification of samples with different composition.