Total internal reflection (TIR)/fluorescence recovery after photobleaching (FRAP), whit-h has been used to study adsorption and surface diffusion of proteins, was modified and applied to study DNA oligonucleotides at liquid/solid interfaces. Conventional TIR/spot FRAP and TIR/pattern FRAP techniques use a photomultiplier tube (PMT) to reveal the adsorption dynamics and surface diffusion rates of biomolecules, respectively. However, they do not provide spatial information on these interfacial processes. in this work, a cooled charge-coupled device camera is substituted for the PMT normally used. Studies of adsorption and surface diffusion of the well-characterized protein bovine serum albumin (BSA) validated the system’s operation. Then, the desorption rate constant for a fluorescently tagged 21-mer DNA oligonucleotide (MW 7140 Da) was determined by spot FRAP. The desorption rate constants for strongly and weakly adsorbed oligonucleotides from (3-aminopropyl)triethoxy silane (APTES)glass were determined to be 0.02 and 0.19 s(-1), respectively. These are of the same order of magnitude as those for ESA (MW 67 000 Da) on APTES glass. The surface diffusion coefficients of oligonucleotide are approximately the same as those for BSA and are dependent on the surface concentration of the molecules on APTES-coated glass. Since the molecules differ by a factor of 10 in molecular weight, these results suggest that the shape of a adsorbate molecule and the strength of adsorbate/substrate interactions play a strong role in interfacial adsorption and diffusion. The substitution of a methyl group in APTES For a hydrogen atom increased the desorption rate constants and surface diffusion coefficients significantly.