In order to investigate the availability of the method using immobilized protein for selection of protein refolding media, two different types of immobilized subtilisin BPN’ were prepared. One was immobilized on agarose gel by the amino groups of the enzyme and the other was immobilized on agarose gel by the carboxyl groups. Using the quantitatively renaturing preparations obtained by repeated denaturation/renaturation treatment of the immobilized subtilisin BPN’, evaluation of various refolding media revealed that the two preparations exhibited similar trends with respect to the refolding yields induced by various media employed and that both of the immobilized subtilisin BPN’ preparations were useful for media selection. Since immobilization can prevent interaction among protein molecules, me propose a selection method of refolding media, especially for refolding of protease, by which media minimizing misfolding are chosen with the immobilized enzyme preparation and the optimal medium for minimizing aggregation and autolysis is chosen from among them with the dissolved enzyme.