In recent years, non-invasive methods for the analysis of biological fluids have attracted growing interest. In this study, we propose a straightforward approach to fabricating silver nanoparticle (AgNP)-coated non-woven polyacrylonitrile substrates for surface-enhanced Raman scattering (SERS). AgNPs were synthesized directly on the substrate using borohydride reduction, ensuring uniform distribution. The optimized SERS substrates exhibited a high enhancement factor (EF) of up to 10⁵ for the detection of 4-mercaptobenzoic acid (4-MBA). To enable glucose sensing, the substrates were further functionalized with glucose oxidase (GOx), allowing detection in the 1–10 mM range. Machine learning classification and regression models based on gradient boosting were employed to analyze SERS spectra, enhancing the accuracy of quantitative predictions (R² = 0.971, accuracy = 0.938, limit of detection = 0.66 mM). These results highlight the potential of AgNP-modified substrates for reliable and reusable biochemical sensing applications.