Unmanned Aerial Vehicle (UAV)-mounted temperature sensors can collect a large amount of valuable 3-dimensional environmental data, especially at locations unsuitable for the installation of traditional weather stations. This paper developed a novel methodology to obtain accurate ambient temperature data using a multi-rotor UAV. Firstly, air temperature sensors and a multi-rotor UAV model were selected based on the objectives of this study. Secondly, field experiments were conducted to determine the influence of in-flight multi-rotor UAV on ambient temperature, to determine the most appropriate locations for the temperature sensor, namely underneath the multi-rotor UAV center and between the landing gears. The data collection, processing, and analysis methods were thus proposed. Thirdly, the accuracy of the proposed methodology was evaluated at a target height by comparing the measured result with that measured by a nearby weather station, and comparing the vertical profile of measured data with the data released by National Environment Agency (NEA) in Singapore. A satisfactory agreement between the two results was observed with an error of between 0 and 0.3 °C. In addition, a case study was conducted to compare ambient temperatures at various heights above different ground materials utilizing the proposed methodology in a park. Besides, the correlation analysis results provided the directions to improve the measurement accuracy.