Liquid temperature measurement is essential for the precise thermal management of micro-heat exchangers, flow chemistry, or microreactor. Thermocouples and RTDs (resistance temperature detectors) cannot meet advanced demands due to the limitation of contact manner. As noninvasive thermometry, laser-induced fluorescence has been widely used for temperature field measurement. However, this method is greatly dependent on the measurement environment: nonuniformity in illumination light intensity, dye concentration distribution, and thickness of the measurement domain including its optical reflection characteristics may strongly affect the measurement result. In this sense, the intensity-based fluorescence technique is not suitable for reliable liquid thermometry, especially in micro/nanoscale because strong nonuniformities can easily occur. In this study, we exploited fluorescence anisotropy for novel microfluidic thermometry to overcome the above-mentioned drawbacks. An advantage of the anisotropic approach as a thermal indicator over the intensity is that the ratiometric measurement while using a single fluorescent molecule. The fluorescent emission under the irradiation by linearly polarized excitation light shows anisotropy, emission intensity in each polarization angle would be different depending on the surrounding temperature. Therefore, measurement of anisotropy in the fluorescent emission leads to evaluating the liquid temperature. We have developed a microscopic liquid temperature measurement system based on fluorescence anisotropy for evaluating the temperature distribution of microfluidic systems. Based on our experimental results, we verified that fluorescence anisotropy can be a promising alternative over conventional laser-induced fluorescence for the advanced thermal management of various microfluidic platforms.