The in-situ heating transmission electron microscopy experiment allows us to observe the time-and temperature-dependent dynamic processes in nanoscale materials by examining the same specimen. The temperature, which is a major experimental parameter, must be measured accurately during in-situ heating experiments. Therefore, calibrating the thermocouple readout of the heating holder prior to the experiment is essential. The calibration can be performed using reference materials whose phase-transformation (melting, oxidation, reduction, etc.) temperatures are well-established. In this study, the calibration experiment was performed with four reference materials, i.e., pure Sn, Al-95 wt%Zn eutectic alloy, NiO/carbon nanotube composite, and pure Al, and the calibration curve and formula were obtained. The thermocouple readout of the holder used in this study provided a reliable temperature value with a relative error of <4%.