Accurate detection of gram-positive bacterial colonies is essential for managing chronic wounds and overcoming delays in healing, as these bacteria can worsen wound conditions and impede recovery. This study introduces a cost-effective electrochemical sensing platform designed to support healthcare professionals in making timely, targeted treatment decisions. We developed the platform using chemically functionalized amine-terminated carbon surfaces combined with the TLR1/TLR2 heterodimer complex to detect gram-positive bacteria. The biosensors featuring these advanced carbon surfaces demonstrated superior performance due to their high surface area and efficient electron transfer capabilities. The TLR1/TLR2-based sensors accurately identified gram-positive bacteria, with a theoretical detection limit of 0.0413 CFU/mL. The sensors also exhibited high selectivity and sensitivity, with a response rate of 220.878 ((ΔR/R)/CFU/mL)/cm2 for the amine-terminated carbon surfaces. This novel electrochemical sensing platform provides an effective solution for real-time detection and management of gram-positive bacterial infections in chronic wound care.