This study utilizes a surface plasmon resonance (SPR) biosensing to investigate the influence of secondary structures on the DNA hybridization and a surface-enhanced Raman scattering (SERS) spectrum to yield analytical data regarding the structure of the oligonucleotides. It is found that the SPR angular shifts associated with the three pairs of 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35°C to 45°C reduces secondary structure effects. On the hybridization with mixture target oligonucleotides, the SPR results demonstrate that secondary structures interfere significantly. Although the kinetics of biomolecular interaction analysis is performed by using SPR sensor, the structural information of the oligonucleotides can not observed directly. The SERS spectrum provides the structural information of the oligonucleotides with silver colloidal nanoparticles adapted as a Raman active substrate. Also, the detection limit of the DNA Raman signal has been successfully improved to reach sub-micro molarity of DNA concentration.