The attachment of biomolecules, in particular proteins, onto solid supports is fundamental in the development of advanced biosensors, bioreactors, affinity chromatographic separation materials, and many diagnostic techniques. In addition, the effective investigation of biomolecular structure and function with scanning probe microscopy often requires a strong attachment of the biomolecule to a substrate. Here, we investigate the binding of the protein catalase to gold surfaces modified by self-assembled monolayers (SAMs). The chemical and physical adsorption of the protein molecules onto SAMs of 8-mercaptopropanoic acid (3-MPA), 11-mercaptoundecanoic acid (11-MUA), and a mixture of the two acid thiols (mixed) was investigated by utilizing tapping mode atomic force microscopy, scanning tunneling microscopy, surface plasmon resonance (SPR), static secondary ion mass spectrometry, and X-ray photoelectron spectroscopy. The surface concentration of catalase adsorbed on the SAMs decreased in the following order : mixed, 11-MUA > 8-MPA. Utilizing the terminal carboxylic acid functionalities, catalase was immobilized with a water-soluble carbodiimide and N-hydroxysuccinimide (NHS). Immobilization resulted in increased coverage of the protein. SPR studies on silver surfaces modified by these SAMs indicate that immobilization of carbodiimide and NHS decreased in the same order, namely mixed > 11-MUA > 3-MPA.