The real-time monitoring of sweat lactate provides valuable physiological insights for assessing exercise outcomes and athletic performance. Conventional lactate detection methods often lack sensitivity, portability, and user-friendliness for in-body applications. To address these limitations, electrochemical biosensing has emerged as a leading approach, enabling non-invasive, real-time analysis. Wearable bioelectronic devices integrate lactate-specific enzymes with electrochemical transducers, offering practical solutions for continuous monitoring. In this study, a wearable lactate electrochemical biosensor was developed using custom screen-printed electrodes modified with a bio-hybrid probe comprising Prussian blue, carbon black, and lactate oxidase. After optimizing key parameters, the biosensor demonstrated a detection limit of 0.06 mM and a linear range up to 20 mM. A filter paper strip was incorporated to enhance sweat collection and serve as the sample chamber, achieving 6% repeatability and efficient sweat handling. The system was validated using three sweat samples, showing strong correlation (96–101%) with LC-MS/MS, a standard laboratory method. The biosensor was integrated into a 3D-printed thermoplastic polyurethane (TPU) armband, designed for efficient sweat collection and transport, combining lightweight durability with a customizable, ergonomic design suitable for dynamic activities. This low-cost, wearable system represents a significant step forward in non-invasive, continuous, and personalized health monitoring, providing a practical tool for tracking physiological parameters in real time.