The paper presents a novel portable electrochemical biosensor for the rapid detection and identification of Gram-positive bacteria, specifically *Staphylococcus aureus* and *Bacillus cereus*. The biosensor leverages the recognition capabilities of vancomycin and aptamers to selectively capture and identify these bacteria. A vancomycin-modified screen-printed carbon electrode (SPCE) was used to capture Gram-positive bacteria, with electrochemical impedance spectroscopy and scanning electron microscopy demonstrating a capture time of 10 minutes and a limit of detection of 2 CFU/mL. The device's potential for aptamer-based bacterial identification was tested using *S. aureus* and *B. cereus* as test strains. The system successfully identified small amounts of these bacteria (100 CFU/mL) in untreated milk or serum within 45 minutes. This biosensor has the potential to serve as an invaluable tool for clinical diagnostics, food safety analysis, environmental monitoring, and security applications, even by inexperienced staff. The study highlights the advantages of using vancomycin and aptamers for rapid, sensitive, and strain-specific detection of Gram-positive bacteria in complex biological matrices.The paper presents a novel portable electrochemical biosensor for the rapid detection and identification of Gram-positive bacteria, specifically *Staphylococcus aureus* and *Bacillus cereus*. The biosensor leverages the recognition capabilities of vancomycin and aptamers to selectively capture and identify these bacteria. A vancomycin-modified screen-printed carbon electrode (SPCE) was used to capture Gram-positive bacteria, with electrochemical impedance spectroscopy and scanning electron microscopy demonstrating a capture time of 10 minutes and a limit of detection of 2 CFU/mL. The device's potential for aptamer-based bacterial identification was tested using *S. aureus* and *B. cereus* as test strains. The system successfully identified small amounts of these bacteria (100 CFU/mL) in untreated milk or serum within 45 minutes. This biosensor has the potential to serve as an invaluable tool for clinical diagnostics, food safety analysis, environmental monitoring, and security applications, even by inexperienced staff. The study highlights the advantages of using vancomycin and aptamers for rapid, sensitive, and strain-specific detection of Gram-positive bacteria in complex biological matrices.