A dual-mode molecular beacon on a multiplexed substrate has been developed and applied to the measurement of unlabeled human viral RNA. The detection system is based on a combined surface-enhanced Raman scattering (SERS) and fluorescent molecular beacon assay that is assembled on Nanobarcodes particles. In this assay, a molecular beacon probe terminated with a fluorescent Raman label dye is conjugated to the metallic Nanobarcodes particles. The molecular beacon probe is a single-stranded oligonucleotide that has been designed with a hairpin structure that holds the dye at 3′-end close to the particle surface when the probe is attached through a 5′-thiol group. In this configuration, the SERS spectrum of the label was obtained and its fluorescence quenched because the dye is in very close proximity to a noble metal surface with nanoscale features (Nanobarcodes particles). The SERS signal decreased and the fluorescence signal increased when target viral RNA was captured by this molecular beacon probe. In addition, a hepatitis C virus reverse transcriptase-polymerase chain reaction (HCV RT-PCR) product was detected using this dual-mode beacon. The development of a multiplexed, label-free assay system with the reassurance offered by detection of two distinctly separate signals offers significant benefits for rapid molecular diagnostics.