Surface-enhanced Raman scattering (SERS)-based biosensors have attracted considerable interest in biomedical applications due to their exceptional sensitivity, non-destructive nature, and inherent multiplexing capability. The effective translation of SERS into bioanalytical platforms relies on strong electromagnetic enhancement from plasmonic nanostructures, coupled with efficient probe–target interactions, to enable trace-level detection of disease-related biomolecules. In this study, we report the fabrication of SERS nanotags composed of gold nanorods (AuNRs) functionalized with Raman reporter molecules and specific targeting ligands for biomarker detection. AuNRs are well suited for SERS biosensing owing to their strong optical absorption and scattering properties. First, AuNR-based SERS nanotags incorporating Raman reporters and cell-specific aptamers were developed for cervical cancer cell detection, demonstrating high selectivity. Second, a highly sensitive and selective strategy for the determination of miR-29a, a cancer-associated microRNA biomarker, was achieved using SERS nanotags in combination with magnetic separation, enabling detection in the picomolar concentration range. Finally, SERS nanotags were constructed for the analysis of glycated human serum albumin (GHSA), a protein biomarker of diabetes mellitus, achieving nanogram-level detection limits. Collectively, these results highlight the strong potential, feasibility, and versatility of AuNR-based SERS biosensors as robust platforms for sensitive and selective biomolecular analysis in medical diagnostics and monitoring.