This study utilizes a surface-enhanced Raman spectroscopy (SERS) based on the attenuated-total-reflection method to investigate the secondary structures of long oligonucleotides and their influence on the DNA hybridization. It is found that the ring-breathing modes of adenine, thyrnine, guanine, and cytosine in Raman fingerprint associated with three 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two 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. The kinetics of biomolecular interaction analysis can be performed by using surface plasmons resonance biosensor, but the structural information of the oligonucleotides can not observed directly. The SERS spectrum provides the structural information of the oligonucleotides with the help of a silver colloidal nanoparticle monolayer by control of the size and distribution of the 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.