<p>Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful plasmonic sensing strategy for rapid, label-free detection of biological targets via localized electromagnetic (EM) field enhancement at metallic nanostructures. In this work, plasmonically active silver nanoparticle (AgNP) aggregate films were fabricated on glass substrates via APTES-assisted surface functionalization followed by immersion coating. The AgNPs were synthesized using a modified Tollens reduction approach employing sucrose as a green reducing agent. Structural and optical characteristics of the resulting plasmonic substrates were investigated by UV–Vis spectroscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM), confirming the formation of densely distributed nanoparticle aggregates capable of generating abundant SERS hotspots. The analytical performance of the substrates was evaluated for the detection of <i>Helicobacter pylori</i>, a clinically significant gastric pathogen. Distinct SERS fingerprints of <i>H. pylori</i> were successfully recorded over a concentration range of 10¹–10¹⁰ Colony-Forming Unit (CFU) mL^− 1. The plasmonic substrate exhibited a limit of detection as low as 10¹ CFU mL^− 1, an empirical enhancement factor (EF) of 4.49 × 10⁶, and excellent signal reproducibility with a relative standard deviation of 6.31% obtained from five independent measurements. The observed sensitivity is attributed to strong localized surface plasmon resonance (LSPR) coupling within AgNP aggregates, resulting in efficient EM enhancement. These findings demonstrate that AgNP aggregate films deposited on glass provide a simple, low-cost, and highly reproducible plasmonic SERS platform for ultrasensitive bacterial detection and molecular fingerprinting, highlighting their potential for rapid biomedical sensing applications.</p> Graphical Abstract <p></p>