We propose a tool to calibrate the coefficient variables of the Steinhart–Hart equation, which are used in temperature measurement with a negative temperature coefficient thermistor. The previous method modifies the coefficient variables manually, but the proposed tool takes a measured temperature and automatically modifies the coefficient variable. The proposed calibration tool provides a graphical user interface program for the convenience of users. It is applied to the 4-point temperature measurement of polymerase chain reaction and has a degree of precision of ±0.1 °C in the temperature measurement evaluation