The reaction results of Polymerase chain reaction (PCR) are highly sensitive to temperature accuracy, but the reaction liquid temperature is hard to be measured directly. In this paper, a virtual temperature sensor is creatively designed to directly obtain the reaction liquid temperature using a model-switching approach. This study first establishes the mathematical models of the temperature control system and obtains precise system models for the heating and cooling stages. Based on this, a virtual sensor is constructed, and a closed-loop fuzzy PID control system is established. Furthermore, the accuracy of the designed virtual temperature sensor is verified through simulation and experimentation. Simulation results show that the fuzzy PID control method has higher temperature accuracy, with a deviation of 0.23 °C, making it more suitable for Peltier systems, as Peltier systems suffer from inconsistent temperature rise and fall models. The experimental results show that the control method using the virtual sensor achieves higher precision in liquid temperature control, with an accuracy of ±0.21 °C. Finally, standard PCR procedures are performed for designed chip, and the results indicate that the temperature sensor and control system designed in this study exhibit significantly higher amplification efficiency, approximately 16 times greater than traditional control methods.