In response to the global need for rapid and precise detection techniques for biomolecules, particularly highlighted by the COVID-19 pandemic, this study explores the application of surface-enhanced Raman spectroscopy (SERS) for precise detection of fluorescent dyes and proteins. Using nanoparticles synthesized via magnetron sputter inert gas condensation, we deposited copper, silver, platinum, gold, and their bimetallic combinations on glass microfiber filters used as 3D substrates. Nanoparticles with sizes ranging from 1.5 to 8 nm were investigated to assess their effect on detection sensitivity. Our results indicate that silver nanoparticles, with an average size of approximately 4–5 nm, perform best among all used metals for SERS, significantly enhancing detection sensitivity. The study successfully demonstrates the effectiveness of SERS in detecting analytes such as rhodamines and bovine serum albumin at very low concentrations (down to 10−9 mol/l for Rhodamine 6G). The findings underscore the potential of SERS for environmental monitoring and diagnostics, highlighting the importance of nanoparticle optimization for achieving high sensitivity, specificity, and reliability in analytical detection.