Mixed self-assembled monolayers (SAMs) to immobilize streptavidin on a gold surface were investigated by measuring the pull-off force between an AFM tip and a biotin-modified surface using CFM. Biotin-LC-NHS was modified on SAMs prepared from a mixed solution of cystamine and MEOH. Increased pull-off forces between the AFM tip and the surface were observed with an increased cystamine mole fraction in the solution. Streptavidin was immobilized onto biotin-LC-NHS modified mixed SAMs and analyzed by tapping AFM. Also, the formation of mixed SAMs containing MUOH and MBDA was confirmed using CFM. The measured pull-off forces on the only MBDA surface were larger than those on the surface with MUOH. These results can be applied to determine an optimal mixing ratio of MUOH and MBDA SAMs that reduces non-specific streptavidin binding onto a surface.