Direct measurement of interaction forces between bovine serum albumin (BSA) adsorbed layers on amino-thiolated gold surfaces has been carried out using colloidal probe atomic force microscopy (AFM). The substrate was prepared via self-assembly of 2-aminoethanethiol on gold in aqueous media. In the absence of BSA, an electric double layer interaction was observed, resulting from protonation of the primary amine groups on the gold surface. Similar electric double layer repulsions were seen in the presence of BSA over a whole range of BSA concentration investigated. These repulsive interactions were found to be dependent on the BSA concentration, that is, the magnitude of the repulsion increases with the BSA concentration. This phenomenon is attributed to the difference in the adsorbed amount of BSA on the amino-thiolated gold surface, which was confirmed using a 27 MHz quartz crystal microbalance (QCM).