To examine the adsorption behavior of antibody fragments (Fab') directly immobilized on a gold surface through S-Au linkage, analyses by surface plasmon resonance (SPR), fluorometry, and atomic force microscopy (AFM) with an excellent blocking technique by the consecutive treatments of longer-poly(ethylene glycol) (PEG) (MW = 5k) and shorter-PEG (MW = 2k), abbreviated as mixed-PEG layer formation, were performed. The results of the SPR analysis suggest that the adsorption-induced inactivation of the antigen-binding activity of Fab' took place gradually on the gold surface, where the activity disappeared almost completely at 60 min after Fab' immobilization. In contrast, in the case of Fab' coimmobilized by the mixed-PEG layer, 70% of the initial antigen-binding activity of the Fab' was retained even 60 min after the construction of the hybrid surface. Using fluorescein-labeled Fab' (FL-Fab'), fluorescence measurement of the constructed surface was carried out. The fluorescence of the FL-Fab' without any blocking agent on the gold surface was gradually quenched and finally decreased to 40% of the initial intensity 60 min after Fab' immobilization. The decrease in the fluorescence intensity is considered to be caused by the change in the distance between the fluorophores labeled on the Fab' and the gold surface, due to the energy transfer from the fluorophores to the gold surface. In contrast, 75% of the initial intensity was observed on the Fab'/mixed-PEG coimmobilized surface. The results obtained from the SPR and fluorometric analyses correlated well with each other; thus, the surface-induced inactivation of the antigen-binding functionality was presumably due to the conformational and/or orientation change of Fab' on the gold surface. AFM studies provided direct information on the time-dependent decrease in the height of the immobilized Fab' on the gold surface. In contrast, the coimmobilization of densely packed mixed-PEG tethered chains around the Fab' on the gold surface suppressed the decrease in the height of Fab', presumably indicating that the conformational and/or orientation change of Fab' was suppressed by the coimmobilized mixed-PEG layer. The new findings obtained in this study are expected to be useful for the improvement of the antibody fragment method and, thus, for the construction of high-performance immuno-surfaces.