In this paper, we report a common intermediate method to present nitrilotriacetic acid (NTA) groups on gold surfaces for immobilizing His-tagged proteins onto the surfaces, and a full characterization of self-assembled monolayers (SAMs) terminating in carboxylic acids [HS(CH2)(15)COOH (C15-COOH), HS(CH2)(11)(OCH2CH2)(3)-OCH2COOH (EG3-COOH), and HS(CH2)(11)(OCH2CH2)(5)OCH2COOH (EG5-COOH)] and coupling reactions of an NTA-containing primary amine [(1S)-N-(5-amino-l-carboxypentyl)iminodiacetic acid; NTA-NH2] with the carboxylic acid on surfaces. The lateral packing densities of the COOH-terminated SAMs were calculated to be 4.32 (for C15-COOH), 3.49 (for EG3-COOH), and 2.65 (for EG5-COOH) molecules/nm(2). The packing densities were decreased by incorporating a relatively flexible ethylene glycol (EG) group into the backbone of alkanethiols and increasing the number of the EG groups in the backbone of alkanethiols. The NTA group was then attached by coupling NTA-NH2 with the COOH group on the surfaces, followed by a Ni(II) complexation. The coupling reaction was characterized by FT-IR spectroscopy, ellipsometry, and XPS, and the coupling efficiency ("yield") was estimated by comparing the experimentally determined N 1s to S 2p (N/S) ratio of XPS data with the N/S ratio calculated for the functionalization of the SAMs presenting NTANi(II): the coupling yields were 30% (for C15-COOH) and 25% (for EG3-COOH and EG5-COOH). Preliminary experiments on the binding of His-tagged proteins onto the surfaces were also performed.