Here we propose a label-free detection of DNA hybridization on Au nanoisland films deposited on Si surfaces by infrared absorption spectroscopy in the geometry of multiple internal reflection (MIR-IRAS). Au nanoisland films were prepared on Si surfaces by evaporation. DNA immobilization via Au-thiol chemistry and subsequent DNA hybridization was monitored with MIR-IRAS. Interaction with complementary DNA produced an IRAS peak at ̃1686cm -1 where the C=O stretching vibration modes of nucleobases appear. On the other hand, addition of non-complementary DNA induced no significant spectral changes in the same region. Sensitive detection of 40nM DNA was achieved owing to the MIR geometry combined with Au nanoisland films deposited on Si which provides low-background absorption around the C=O stretching vibration region. Comparison between experimental results and theoretical prediction, obtained using the ab initio calculation, was also examined.