This study presents a simple electrochemical DNA biosensor to accurately detect Klebsiella pneumoniae DNA. The biosensor uses glycine functionalized iron oxide nanoparticles (glycine@Fe3O4) to capture Klebsiella pneumoniae DNA. The as-synthesized nanoparticles were characterized using X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy, and transmission electron microscopy, and created a sensitive electrode surface that produced strong electrochemical signals when DNA attached. Electrochemical techniques, including cyclic voltammetry and square wave voltammetry, were used to develop a biosensor for detecting Klebsiella pneumoniae DNA. The biosensor showed limits of detection of 3.27 nM in the 30-90 nM range and 3.94 nM in the 120-270 nM range, with limits of quantification of 3.90 nM and 11.95 nM, respectively. The sensitivity, determined from calibration curves, was 0.1009 μA nM-1 for the low range and 0.0838 μA nM-1 for the high range. The biosensor demonstrated high sensitivity and selectivity, effectively distinguishing the analyte from interferents like albumin and folic acid. Molecular docking showed strong DNA binding (-6 kcal mol-1). Lab tests confirmed detection of Klebsiella pneumoniae antimicrobial resistance genes (SHV, TEM, CTX-M, OXA-1) via PCR-based gel electrophoresis. This easy-to-use non-enzymatic biosensor offers fast, accurate, rapid, sensitive, and specific Klebsiella pneumoniae DNA detection, valuable for point-of-care diagnostics and antimicrobial resistance monitoring.