This study utilizes a nanoplasmon-enhanced Raman scattering based on the attenuated-total-reflection (ATR) 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, thymine, 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 ATR-Raman spectrum can provide the structural information of the oligonucleotide monolayer on the sensing surface with the help of a silver patterned nanostructure film based on the finite-difference time-domain simulation and the e-beam lithography fabrication adapted as an ATR-Raman active substrate.