This study presents a novel electrochemical biosensor for the detection of Helicobacter pylori VacA protein using a graphene-Au nanocomposite platform. The nanocomposite was synthesized via a one-pot reduction method and characterized using SEM, XRD, and FTIR. The biosensor fabrication process was monitored through cyclic voltammetry and electrochemical impedance spectroscopy. Optimization studies revealed ideal conditions of pH 7.4, 30-minute incubation time, and 100 μg/mL antibody concentration. The biosensor exhibited a linear response range of 0.1–100 ng/mL with a detection limit of 0.03 ng/mL. Selectivity tests against common interfering substances showed minimal cross-reactivity (<5 % signal change). The sensor demonstrated good reproducibility (3.8 % RSD) and stability (95 % signal retention after 30 days). In spiked serum samples, recoveries ranged from 96.5 % to 103.2 % with RSDs below 4.5 %. Compared to traditional methods, this biosensor offers improved sensitivity and shorter analysis time for VacA detection, potentially enhancing early diagnosis of H. pylori infections and monitoring treatment efficacy.