The widespread use of antibiotics in livestock and poultry leads to antibiotic residues in food, posing public health risks. To ensure food safety, monitoring antibiotic levels in dairy and poultry is essential, especially for Penicillin-G (Pen-G), a frequently used β-lactam antibiotic. This study presents an electrochemical aptasensor for detecting Pen-G in food samples, using chemically reduced graphene oxide (crGO) conjugated with Pen-G-specific aptamer on electrode. The sensors construction was validated via various microscopy and spectroscopy techniques and its performance optimized by adjusting factors such as pH, scan rate, temperature, concentration of aptamer, and reaction time using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). It achieved a detection limit of 1.24 pM, effectively distinguishing Pen-G from other antibiotics in real milk, meat, and egg samples, with stability for up to 3 weeks, making it a valuable tool for antibiotic monitoring in food products.