This paper illustrates an application of a technique for predicting the thermal characteristics of a bidirectional thermocycling device for polymerase chain reaction (PCR). The micromilling chamber is oscillated by a servo motor and contacted with different isothermal heating blocks to successfully amplify the DNA templates. Because a comprehensive database of contact resistance factors does not exist, it causes researchers to not take thermal contact resistance into consideration at all. We are motivated to accurately determine the thermal characteristics of the reaction chamber with thermal contact effects existing between the heater surface and the chamber surface. Numerical results show that the thermal contact effects between the heating blocks and the reaction chamber dominate the temperature variations and the ramping rates inside the PCR chamber. However, the influences of various temperatures of the ambient conditions on the sample temperature during three PCR steps can be negligible. The experimental temperature profiles are compared well with the numerical simulations by considering the thermal contact conductance coefficient which is empirical by the experimental fitting. To take thermal contact conductance coefficients into consideration in the thermal simulation is recommended to predict a reasonable temperature profile of the reaction chamber during various thermal cycling processes. Finally, the PCR experiments present that Hygromycin B DNA templates are amplified successfully. Furthermore, our group is the first group to introduce the thermal contact effect into theoretical study that has been applied to the design of a PCR device, and to perform the PCR process in a bidirectional thermocycler.