A facile and sensitive glucose sandwich assay using surface-enhanced Raman scattering (SERS) has been developed. Glucose was captured by 3-aminopheyonyl boronic acid (APBA) modified Ag nanoparticles decorated onto a polyamide surface. Then, Ag nanoparticles modified with 3-amino-6-ethynylpicolinonitrile (AEPO) and APBA were used as SERS tags. APBA forms specific cis-diol compounds with glucose molecules avoiding interference by other saccharides and biomolecules in urine enabling its selective detection. As the actual Raman reporter, AEPO exhibited a distinctive SERS peak in the Raman silent region, thus increasing the sensitivity of the glucose detection to 10-11 M. Additionally, the developed SERS assay was reusable, and its applicability in artificial urine samples demonstrated future clinical utility confirming the potential of this innovative technology as a diagnostic tool for glucose sensing.