We present a DNA biosensor based on self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) molecules adsorbed on gold surfaces. Previous works have shown that PNA molecules at an optimal concentration can be self-assembled with their molecular axes normal to the surface. In such structural configuration BioSAMs of PNAs maintain their capability for recognizing complementary DNA. We describe the combined use of PM-RAIRS and synchrotron radiation XPS for the detection and spectroscopic characterization of PNA-DNA hybridization process on gold surfaces. RAIRS and XPS are powerful techniques for surface characterization and molecular detection, which do not require a fluorescence labeling of the target. We present a characterization of the spectroscopic IR and XPS features, some of them associated to the phosphate groups of the DNA backbone, as an unambiguous signature of the PNA-DNA heteroduplex formation. The N(1s) XPS core level peak after DNA hybridization is decomposed in curves components, and every component assigned to different chemical species. Therefore, the results obtained by means of two complementary structural characterization techniques encourage the use of PNA-based biosensors for the detection of DNA molecules on natural samples.