Precise temperature regulation is a crucial guarantee for many microfluidic analyses. This study presents a linear cooling method of microfluidic chips based on a single TEC, which can achieve synchronous observation of samples. A numerical model was developed and experimentally verified to analyze the temperature responses under different driving current mechanisms of TEC. Based on simulation and experimental analysis, this study proposes to achieve linear cooling based on TEC driven by polynomial function current mechanism. The implementation process is clarified, and the iterative method to obtain the polynomial function current mechanism is provided and elaborated. Using a commercial TEC, the linear cooling of the microfluidic chip from 25.2 °C to −19.7 °C was successfully achieved through both simulation and experiment. The linear cooling rates ranging from 24 °C/min to 41 °C/min with linearity higher than 0.998 were obtained. Moreover, the influencing factors of linear cooling were discussed. It is found that the temperature of the TEC hot side has a significant impact on the linear cooling of the sample cell, while the impact of nitrogen gas temperature is almost negligible. Results also indicate that both the minimum and maximum cooling rates increase as TE-element length increases and TE-element height decreases.