A Langmuir monolayer of carbonic anhydrase (CA) unfolded at an air/water interface was transferred onto the hydrophobic surface of a silicon wafer by means of the Langmuir-Schaefer technique. The transferred CA film was biotinylated and was incubated in a streptavidin (SAv) solution to obtain a densely packed SAv layer by biotin-SAv linkage. Biotinylated proteins including ferritin, catalase, alcohol dehydrogenase, and carbonic anhydrase were incubated with the SAv layer and binding of these proteins was examined by atomic force microscopy. High-density binding of the biotinylated proteins was observed, whereas the amount of adsorbed non-biotinylated proteins was low or negligible. The SAv layer on the Langmuir-Schaefer film of unfolded protein could become a basic architecture for protein immobilization studies. (C) 2016 Elsevier B.V. All rights reserved.