In this study, we have firstly investigated the effects of dipping time, solution concentration and solvent types on the formation of self-assembled monolayers (SAMs) with 3-mercaptopropyltrimethoxysilane (MPTS) molecules on the Si(0 0 1) surfaces. Ellipsometric measurements showed that monolayers with a thickness of about 0.73 nm were formed when the dipping time is about 1 h, while more profound agglomerations were observed for longer time periods and MPTS solutions with higher concentrations. Monolayers were formed with solvents having larger dielectric constants. Contact angle measurements were in good correlation with the ellipsometric data. 5′-Thiolated oligodeoxynucleotides (ODNs) probes were immobilized onto these thiol-terminated SAMs by disulfide bond formation. The thickness of the ODN-layer on the MPTS modified surfaces reached almost a constant value of about 2.5 nm in 1 h. Target ODNs were detected by monitoring hybridization onto the surfaces by ellipsometry. The analytical signal (the delta (Δ) value) measured was correlated with the target ODNs concentration.