This paper reports a low-cost microthermostat that is able to maintain a constant temperature necessary for restriction enzyme digestion. Polydimethylsiloxane (PDMS) and Pyrex glass were used to make the microthermostat, because PDMS is a cheap and mass-producible material and both PDMS and glass have very good biocompatibility compared to the more commonly used silicon. A heater made of Au wiring patterned on Pyrex glass was used to control the temperature. A PDMS replica molding technique was used to fabricate a reaction chamber with 3.6 μl capacity. Restriction enzyme digestion was performed by using the fabricated microthermostat and by a conventional method. Then, using gel electrophoresis, we compared results between the microthermostat and conventional methods. It was found that restriction enzyme digestion using the microthermostat required 5 min of heating.