This research reports a multifunctional thermal biosensor that has thermocouple structures integrated with freestanding microfluidic channels for bio-sensing based on enzyme-catalyzed reactions. The free-standing SU-8 polymer-based microfluidic measurement chamber allows sensitive measurement of small volumes of liquid samples and reduces heat loss to improve the sensitivity. The fabricated thermocouple biosensor is capable of enzyme-catalyzed reaction detection for healthcare applications. Reactive enzymes are immobilized on microspheres packed in the chambers. The fabricated thermocouple biosensor shows a sensitivity of approximately 0.5 V/W and a thermal time constant of less than 100ms. The Seebeck-coefficient of thermocouple biosensor obtains a sensitivity of 1.1mV/°C with the measurement of IR thermometry. The results demonstrate that the sensor was capable of enzyme-catalyzed reaction detection for healthcare application.