Mixed self-assembled monolayers of biotinylated-and ethylene glycol-terminated long-chain alkanethiols were prepared on gold surfaces in an attempt to develop a reliable protocol for immobilization of streptavidin. A broad range of surface analytical techniques including ellipsometry, atomic force microscopy, and infrared, fluorescence, and X-ray photoelectron spectroscopy were used to characterize the SAMs before and after immobilization of streptavidin. The first part of the work was focused on finding the mixing conditions that lead to optimum binding capacity of streptavidin. Mixed SAMs prepared from loading solutions containing 75-95% of the biotinylated alkanethiol resulted in high immobilization levels of functional streptavidin. The thin layers of streptavidin subsequently can be used for the immobilization of a broad spectrum of biotinylated biomolecules (e.g. oligonucleotides, cDNA, peptides, proteins, antibodies, and carbohydrates) and provides therefore an excellent platform for the fabrication of chips/arrays for biosensor and screening applications. This is successfully demonstrated by monitoring the hybridization between a biotinylated 24-mer capturing oligonucleotide and a labeled target 89-mer DNA using a fluorescencebased DNA-microarray detection system. Moreover, the DNA-microarray experiments also revealed (i) good selectivity when comparing the response of the complementary oligonucleotide with that of a random 24-mer capturing oligonucleotide and (ii) low levels of nonspecific binding to the streptavidin surface.