In the analysis of high resolution melting (HRM) of microfluidic fluorescence PCR amplification products, fluorescence intensity has a sudden change with the delink of DNA chain, while whether the abrupt change can be detected is dependent on the accuracy of temperature control. A kind of multi-threshold PID algorithm was put forward, and a temperature control system based on FPGA was designed to adapt to the analysis of high resolution melting. Based on the difference of the measurement value and the set value, the temperature change can be controlled accurately through changing the pulse-time ratio of pulse width modulation (PWM) wave and the control frequency of the algorithm from the FPGA circuit system. After temperature calibrations of the measurement system, the accuracy precedes 0.1℃ and the resolution achieves ±0.01℃. The result of HRM experiment shows that this system can detect the single base mutation of DNA sequence successfully, and the temperature control system can meet the design requirements.