The accurate determination of melting temperature (Tm) of oligonucleotide hybridization is the primary for various biosensors based on the technique of using surface-tethered nucleic acids as probes. In this paper, a new type of nanosensor based on the reflectometric interference spectroscopy (RIFS) was proposed to determine the Tm in real time. A composite solid nanostructure, i.e., a nanoporous anodic alumina film with a 15 nm top layer of gold sputtered on surface, was employed as the substrate of the RIFS-based nanosensor, and a 30 bp oligonucleotide molecule was bond to the inner wall of pores and hybridized, and then its Tm was detected with raising temperature. The experimental data showed that there were obvious signs of dsDNA molecules denatured and melted into ssDNA molecules at 95 , which was highly consistent with the theoretical analysis. The cost-effective RIFS nanosensor has excellent thermal and physicochemical stabilities, higher determination accuracy, and great potential in the field of biomolecule research. © 2019 Elsevier GmbH