A sulfanyl-terminated poly(ethylene glycol) (PEG)-brushed layer was constructed on a gold sensor platform by consecutive treatment with a sulfanyl-ended semitelechelic PEG (2 kDa, hereafter "MeO-PEG-SH (2k)") and a sulfanyl-ended telechelic PEG (5 kDa, hereafter "SH-PEG-SH (5k)"). Our strategy of constructing the sulfanyl-terminated PEG-brushed gold Surface is based on mixed-PEG-brush formation from the longer SH-PEG-SH (5k) and the shorter MeO-PEG-SH (2k), where the preimmobilized shorter MeO-PEG-SH (2k) prevents loop formation in the longer SH-PEG-SH (5k) on the surface and the free sulfanyl group at one end of the longer SH-PEG-SH is exposed to the mixed-PEG tethered-chain surface. From the experimental results obtained from surface plasmon resonance analysis, it became apparent that the immobilization density and the orientation of the longer SH-PEG-SH (5k) on the gold surface Could be controlled by the amount of preimmobilized shorter MeO-PEG-SH (2k). Under the optimized conditions of MeO-PEG-SH (2k) premodification, the constructed MeO-PEG-SH (2k)/SH-PEG-SH (5k) mixed layer conjugated efficiently with the maleimide-instal led proteins and the antibody Fab' fragments, accompanied by an appreciable nonfouling characteristic against bovine serum albumin as strong as that of the MeO-PEG-SH (5k)/MeO-PEG-SH (2k) mixed surface, which was reported in our previous work; it also showed a superior nonfouling characteristic compared to the commercially available carboxymethylated dextran surface (Uchida. K.; et al. Biointerphase 2007, 2 (4), 126-130). Furthermore, from the experimental results of the X-ray photoelectron spectrometry analysis, the presence of both Au-bound and Au-unbound sulfur species was confirmed on the SH-PEG-SH (5k)/MeO-PEG-SH (2k)-modified gold surface. These results clearly indicate that the preimmobilized shorter MeO-PEG-SH (2k) not only increased the nonfouling characteristic of the PEG tethered-chain surface but also prevented loop formation in the longer SH-PEG-SH (5k) on the gold surface. Since the protein-installed SH-PEG-SH (5k)/MeO-PEG-SH (2k)modified surface showed a strongly nonfouling characteristic and recognized the target molecules selectively, this new mixed-brush-formation technique using longer sulfanyl-ended telechelic PEGs and shorter semitelechelic PEGs is a simple yet effective method of constructing a strongly nonfouling terminal-functionalized gold surface for protein immobilization.