BACKGROUND: Polyethyleneimine (PEI) has attracted wide interest due to not only its good hydrophilicity but also its high density of amino groups across the polymer chain. In order to study in depth the effect of PEI as spacer arm on dye ligand modification and further protein adsorption, poly(methylmethacrylate) (PMMA) magnetic microspheres were fabricated, grafted with PEI, and further modified with an affinity dye-ligand, Cibacron Blue F3GA (CB). RESULTS: CB density of these CB-PEI-PMMA microspheres was determined to be 370.7 mu molg(-1) for CB-PEI600-PMMA, 355.2 mu mol g(-1) for CB-PEI1800-PMMA, and 317.6 mu molg(-1) for CB-PEI70000-PMMA microspheres, respectively. Moreover, bovine serum albumin (BSA) adsorption capacity of PEI-PMMA and CB-PEI-PMMA microspheres increased greatly compared with PMMA microspheres. The effects of temperature, BSA initial concentration, pH and ionic strength on BSA adsorption were studied. CB-PEI 70000-PMMA microspheres exhibited the maximum adsorption of 103.6 mg g(-1). CONCLUSION: The results demonstrated the versatility of PEI as spacer arm. The information obtained from the present work could be utilized for rational design of carriers for protein adsorption or enzyme immobilization. (C) 2017 Society of Chemical Industry.