In this study, human serum albumin (HSA) adsorption properties of reactive green HE-4BD-attached monodisperse poly(styrene-2-hydroxyethyl methacrylate) [poly(St-HEMA)] microspheres were investigated. Poly(St-HEMA) microspheres with an uniform size of 4.0 mum in diameter were produced by the dispersion copolymerization of St and HEMA in an ethanol-water medium. Reactive green HE-4BD was covalently attached onto the poly(St-HEMA) microspheres via a nucleophilic substitution reaction between the hydroxyl groups of HEMA and triazinyl chloro groups of dye with the equilibrium coupling capacity of 45.8 mumol dye (g polymer)(-1). The poly(St-HEMA) monodisperse microspheres were characterized by scanning electron microscopy. The effect of concentration of HSA, medium pH, and ionic strength on the adsorption efficiency of dye-attached microspheres were studied in a batch system. The nonspecific adsorption of HSA on the poly(St-HEMA) microspheres was 2.2 mg g(-1). Reactive green HE-4BD attachment significantly increased the HSA adsorption up to 221 mg g(-1). The langmuir adsorption model was found to be applicable in interpreting HSA adsorption by reactive green HE-4BD attached microspheres. Significant amount Of the adsorbed HSA (up to 92%) was eluted in 1 hr in the elution medium containing 1.0-M NaCl. To determine the effects of adsorption conditions on possible conformational changes of HSA structure, fluorescence spectrophotometry was employed. We resulted that poly(St-HEMA) dye-affinity microspheres can be applied for HSA adsorption without causing any significant conformational changes. Repeated adsorption/ elution processes showed that these dye-attached monodisperse microspheres are suitable for HSA adsorption.