Detection and estimation of various biomolecular samples are often required in research and clinical laboratory applications. Present work demonstrates the functioning of a surface‐enhanced Raman scattering (SERS) substrate that has been obtained by drop‐casting of citrate‐reduced gold nanoparticles (AuNPs) of average dimension of 23 nm on a bare blu‐ray digital versatile disc (BR‐DVD) substrate. The performance of the proposed SERS substrate has been initially evaluated with standard Raman active samples, namely malachite green (MG) and 1,2‐bis(4‐pyridyl)ethylene (BPE). The designed SERS substrate yields an average enhancement factor of 3.2 × 106 while maintaining reproducibility characteristics as good as 94% over the sensing region of the substrate. The usability of the designed SERS substrate has been demonstrated through the detection and analysis of purified rotavirus double‐stranded RNA (dsRNA) samples in the laboratory environment condition. Rotavirus RNA concentrations as low as 10 ng/μL could be detected with the proposed sensing scheme. A highly sensitive, low‐cost surface‐enhanced Raman scattering (SERS) substrate has been developed for detection and analysis of the Raman spectrum of rotavirus RNA by exploiting the patterned nanochannels of a commercially available blu‐ray digital versatile disc (BR‐DVD) as substrate. Gold nanoparticles of dimension comparable to the depth of the nanochannels generate enhanced hot‐spot regions in combination with guided‐mode resonance field. The proposed SERS substrate yields a high enhancement factor along with a good degree of reproducibility of the scattered Raman signal.