In the present work, for noninvasive measurement of the liquid temperature in microchannels, the two-color ratiometric laser-induced fluorescence (LIF) technique was combined with the confocal microscopy. By using this technique, the fluorescent light from the tiny volume around a focusing spot can be selectively detected, and it enables us to measure the local liquid temperatures even at the close vicinity of the walls. To check the general performance of this method, as the preliminary stage, a test section consisting of two horizontal plates in different temperatures, separated by a narrow gap filled with a mixture of rhodamine B (a temperature-sensitive dye) and methanol was made, and the temperature distribution was examined. Based on the relationship between the fluorescence intensity and the temperature, a linear temperature distribution across the gap (by conduction heat transfer) could be confirmed. However, the measured results were subject to external disturbances such as the excitation laser intensity fluctuation and the irregular reflection of the light from the glossy walls. Therefore, in the second stage, rhodamine 110 (a temperature-insensitive dye), having a different emission spectrum peak (520 nm) from the rhodamine B (575 nm), was added to the mixture. In principle, the external disturbance effects cancel out each other when the intensity ratio between rhodamine B and rhodamine 110 is considered (instead of taking data only with rhodamine B). To compensate a substantial reduction in the fluorescence intensity from rhodamine 110 by the re-absorption phenomenon within the liquid, which is inherent in using the two-color thermometry, dependency of the intensity ratio on the depth of the measuring point was examined as well. In summary, the two-color ratiometric confocal-LIF thermometry was found to be a very useful tool in measuring the local temperatures of the liquid flow field in microfluidic devices.