We present the use of thiol-modified and phosphorothioate (PT)-modified oligonucleotides for building DNA microarrays on the gold surface of surface plasmon resonance (SPR) chips. PT-modified oligonucleotides (PTOs) have several advantages in comparison to thiol-modified ones. They do not form disulfides and the PT groups can be introduced in any desired position of the molecular backbone of the oligonucleotide. Additionally, modifications with PT groups are not as cost-intensive as thiol groups. The direct immobilization of the oligonucleotides via thiol and PT groups is compared. The affinity of the PT groups to gold is lower than that of thiol groups. Nevertheless, the hybridization kinetics of a model polymerase chain reaction (PCR) product could be studied in real time on a DNA microarray for both types of modified oligonucleotides. This contributes to clarification of previous, contradictory reports on the use of PTOs for their attachment to gold surfaces. The immobilization of PTOs could be improved by the introduction of iodoacetylated surfaces, which are reactive to bind PTs as well as thiols with high efficiency. Furthermore, the influence of the probe structure on the probe density and the hybridization was investigated.