This study presents a novel electrochemical aptasensor for the rapid detection and identification of vancomycin-sensitive Gram-positive bacteria. The sensor utilizes vancomycin-modified screen-printed carbon electrodes (SPCEs) to selectively capture Gram-positive bacteria, followed by aptamer-based identification. The sensor was tested with Staphylococcus aureus and Bacillus cereus, achieving detection limits of 2 CFU/mL in 10 minutes. The system demonstrated the ability to identify these bacteria in untreated milk and serum within 45 minutes. The sensor's performance was evaluated using electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM), revealing a significant increase in charge transfer resistance (Rct) with higher bacterial concentrations. The aptamer-based identification step allowed for the differentiation of bacterial strains, with specific aptamers binding to target bacteria and causing measurable changes in current intensity. The sensor's results showed high sensitivity, reproducibility, and potential for use in various applications, including clinical diagnostics, food safety, and environmental monitoring. The study highlights the advantages of this biosensor over traditional methods, offering a rapid, sensitive, and cost-effective solution for bacterial detection.This study presents a novel electrochemical aptasensor for the rapid detection and identification of vancomycin-sensitive Gram-positive bacteria. The sensor utilizes vancomycin-modified screen-printed carbon electrodes (SPCEs) to selectively capture Gram-positive bacteria, followed by aptamer-based identification. The sensor was tested with Staphylococcus aureus and Bacillus cereus, achieving detection limits of 2 CFU/mL in 10 minutes. The system demonstrated the ability to identify these bacteria in untreated milk and serum within 45 minutes. The sensor's performance was evaluated using electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM), revealing a significant increase in charge transfer resistance (Rct) with higher bacterial concentrations. The aptamer-based identification step allowed for the differentiation of bacterial strains, with specific aptamers binding to target bacteria and causing measurable changes in current intensity. The sensor's results showed high sensitivity, reproducibility, and potential for use in various applications, including clinical diagnostics, food safety, and environmental monitoring. The study highlights the advantages of this biosensor over traditional methods, offering a rapid, sensitive, and cost-effective solution for bacterial detection.