It is generally known that both electrostatic and hydrophobic interactions are major driving forces for protein adsorption at solid/liquid interfaces, but relatively little is known about how these interactions affect the interfacial behavior of single-stranded DNA molecules. This information is important for the design of DNA hybridization sensors. In this study, total internal reflectance microscopy/fluorescence recovery after photobleaching (TIR/FRAP) was applied to measure the desorption rate constants and the surface diffusion coefficients of a 21-base oligonucleotide on four different surfaces including two cationic amino-silanized and two hydrophobic-silanized surfaces. Adsorption isotherms of the oligonucleotide were determined by using porous glass beads that were identically silanized. The results indicate that the oligonucleotide adsorbs reversibly and interacts strongly with the four surfaces studied. Approximately 50% less oligonucleotide adsorbed on the hydrophobic substrates than on cationic amino-silanized glasses. The desorption rate constant decreases as the density of the hydrophobic silane surface layer increases and it depends on the chemical properties of the substrate. The surface diffusion coefficients depend on the density of the adsorbed oligonucleotides on the hydrophobic surfaces in phosphate-buffered saline (pH 7.4) solution (PBS). When ethanol was added to the adsorption buffer, the initial slopes of the adsorption isotherms for hydrophobic dimethyldichlorosilane-treated (DMS) surface as well as the two secondary amino-silanized glasses were lowered. On the other hand, the kinetics of adsorption and surface diffusion of oligonucleotides in the presence of ethanol on all substrates was independent of the substrate properties and the equilibrium adsorption affinity. DNA oligonucleotides have both ionic and hydrophobic characteristics which result in complex adsorption and surface diffusion behavior on glass silanized with a range of ionic and hydrophobic silanes. The orientation of the oligonucleotide lying on the surface and the quantity adsorbed appear to be influenced by whether the surface is cationic or hydrophobic.