An integrated thermal biosensor array was designed and fabricated for the simultaneous determination of glucose, urea and penicillin. The sensor contains six thin-film thermistors which were fabricated on quartz chip by micromachining and semiconductor technology. This sensor allows the determination of three different analytes nearly simultaneously by serially partitioning a single microchannel into three distinct detection regions. Each of the regions contained one immobilized enzyme matrix and a pair of thermistors for differential measurement of temperature changes in association with the enzyme reactions. Using this device, glucose, urea and penicillin-G at different concentrations were determined in the mixed samples. Linear ranges of 1 to at least 25 mM urea and penicillin, and up to 20 mM glucose were obtained using a sample volume of 20 μL.