Detection of bacterial pathogens including Staphylococcus aureus from the blood samples of sepsis patients is challenging due to the lack of fast and sensitive diagnostic methods. Hence, the development of a simple, rapid, accurate, and portable platform for S. aureus detection is necessary to ensure accurate diagnosis, treatment, and infection control in clinical settings. This paper presents a simple method in fabricating a label-free electrochemical aptasensor on a printed circuit board gold electrode for S. aureus detection. Briefly, an S. aureus-specific aptasensor was fabricated by modifying the surface of a gold electrode with functionalized multi-walled carbon nanotubes (f-MWCNTs) to improve the electrical conductivity and detection sensitivity. The aptamer-functionalized electrode was then characterized using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The surface morphology of the modified electrode was observed using Field-Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-ray Spectroscopy (EDX) analysis. The result shows that the aptasensor platform can successfully detect 2×103 S. aureus cells using only a 2 µL-volume of bacterial suspension. In conclusion, the label-free electrochemical aptasensor presents a promising approach for further development of a simple, robust, and sensitive assay for the diagnostic application of S. aureus infection.