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In this article, experimental and numerical analyses to investigate the thermal control of an innovative vortex tube based polymerase chain reaction (VT-PCR) thermocycler are described. VT-PCR is capable of rapid DNA amplification and real-time optical detection. The device rapidly cycles six 20 μl 96 bp λ-DNA samples between the PCR stages (denaturation, annealing, and elongation) for 30 cycles in approximately 6 min. Two-dimensional numerical simulations have been carried out using computational fluid dynamics (CFD) software FLUENT v.6.2.16. Experiments and CFD simulations have been carried out to measure/predict the temperature variation between the samples and within each sample. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the temperature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation around ±0.5 and 1.6 °C, respectively). During cooling, by adjusting the cold release valves in the VT-PCR during some stage of cooling, the heat transfer rate has been controlled. Improved thermal control, which increases the efficiency of the PCR process, has been obtained both experimentally and numerically by slightly decreasing the rate of cooling. Thus, almost uniform temperature distribution between and within the samples (within 1 °C) has been attained for the annealing stage as well. It is shown that the VT-PCR is a fully functional PCR machine capable of amplifying specific DNA target sequences in less time than conventional PCR devices.