question: We developed a dual-mode surface-enhanced Raman scattering (SERS)-based aptasensor that can accurately diagnose and distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/H1N1 at the same time. Herein, DNA aptamers that selectively bind to SARS-CoV-2 and influenza A/H1N1 were immobilized together on Au nanopopcorn substrate. Raman reporters (Cy3 and RRX), attached to the terminal of DNA aptamers, could generate strong SERS signals in the nanogap of the Au nanopopcorn substrate. Additionally, the internal standard Raman reporter (4-MBA) was immobilized on the Au nanopopcorn substrate along with aptamer DNAs to reduce errors caused by changes in the measurement environment. When SARS-CoV-2 or influenza A virus approaches the Au nanopopcorn substrate, the corresponding DNA aptamer selectively detaches from the substrate due to the significant binding affinity between the corresponding DNA aptamer and the virus. As a result, the related SERS intensity decreases with increasing target virus concentration. Thus, it is possible to determine whether a suspected patient is infected with SARS-CoV-2 or influenza A using this SERS-Based DNA aptasensor. Furthermore, this sensor enables a quantitative evaluation of the target virus concentration with high sensitivity without being affected by cross-reactivity. Therefore, this SERS-based diagnostic platform is considered a conceptually new diagnostic tool that rapidly discriminates against these two respiratory diseases to prevent their spread.