Among the different analytical techniques, surface-enhanced Raman scattering (SERS) approach is a widely used technique for the detection and analysis of various chemicals and biological samples. Present study reports a low- cost, sensitive SERS substrate that has an ability to detect rotavirus in clinical stool samples. The proposed SERS substrate has been fabricated through drop-casting of silver nanoparticles (AgNPs) on a printing-grade paper. Rotavirus particles were extracted from clinical stool samples. The presence of rotavirus antigen in stool samples was confirmed using enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and sequencing. The characteristic Raman peaks of rotavirus (RV) particles in solution were found to be significantly enhanced when Raman signals were recorded from the paper-based SERS substrates. Using the proposed SERS substrate, rotavirus samples with concentration as low as 1% could be reliably recorded by the Raman spec- trometer. The paper SERS substrate reported herein is an extremely cost-efficient platform and may find ap- plications in other research and clinical laboratories as well.