The surface properties of recombinant microbial transglutaminase (MTG) were characterized by varying the pH through the aqueous two-phase system (ATPS) method in order to select the effective refolding pathways. It was found that the MTG activity was lost and intrinsic tryptophan fluorescence was varied at pH values less than 3.8. In this pH region, the surface net charge and surface net hydrophobicity of the MTG, determined by the ATPS method, gradually increased. At around pH 3.5, the local hydrophobicity of the MTG was found to be maximal.