The temperature measurement of liquids is essential in many fields such as the thermal management of a chemical process or material synthesis. It is relatively easy to measure the liquid temperature on the macroscale, but temperature imaging on the micro/nanoscale is still challenging. Conventional methods such as the use of thermocouples and resistance temperature detectors and laser-induced fluorescence have drawbacks when applied to microfluidic temperature imaging techniques. In the present work, fluorescence anisotropy (FA) was used as a liquid temperature measurement method on the microscale. FA has an advantage over conventional methods of intrinsic normalization of the light intensity, which enables ratiometric measurement even when using a single wavelength from a fluorophore. We measured FA values in liquids of different viscosities and temperatures using a spectrofluorometer having two rotational polarizers, then obtained the temperature sensitivity of FA. The temperature sensitivity of FA was also theoretically investigated using the derivative of Perrin’s equation, which relates FA, viscosity, temperature, fluorescence lifetime, and molecular size. The experimental results show that each molecule has an optimal viscosity range indicating the maximum temperature sensitivity and that fluorescein isothiocyanate–dextran conjugates with smaller molecular weights have higher sensitivity. Also, reasonable agreement between experimental and theoretical results was confirmed. Consequently, it was clarified that the temperature sensitivity of FA can be controlled by labeling to adjust the required viscosity range of the sample solution and that theoretical estimation provides qualitative guidance for FA-based thermometry.