We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.