A microfluidic biosensor for real-time monitoring of lactic acid (LA) contained in sweat was fabricated and tested. Sweat is one of the most promising body fluids for non-invasive assessment of biological components reflecting physiological condition. In order to measure sweat LA continuously, we developed a microfluidic biosensor, which is highly specific with LA, and a sampling element that transports whole secretions to the biosensor. In this study, the biosensor was first fabricated and its fundamental characteristics were investigated. The biosensor showed sufficient sensitivity (0.16 nA μM -1 ) to measure sweat LA and high specific activity suitable for the sweat monitoring purpose was also confirmed. Real-sample test was also carried out using our system. The microfluidic biosensor was attached on the upper arm of healthy male subject. Sweat LA was continuously monitored during bike exercise. As a result, elevation of LA was successfully detected (62.4 μg/cm 2 /min for anaerobic exercise), and the signal decreased to the background level after the exercise. This possibility of non-invasive evaluation of metabolic activity was demonstrated.