“2-Color laser-induced fluorescence” (2-C LIF) is a novel quantitative temperature measurement method. This can eliminate the undesirable spatio-temporal fluctuation of excitation intensity occurred by light source and channel configuration using a ratio-metric technique with two fluorescence images. Therefore, it can obtain quantitative temperature distribution, and thus simplify temperature calibration in micro channel. In this paper, we applied it to micro-electrokinetic flow. Consequently, the spatial intensity error could be reduced to about 95% as compared with 1-Color LIF image. The temperature distribution in microchannel was quantitatively visualized at the high resolution 0.12×0.12 μm, high sensitivity 1.98%/K and high accuracy ± 0.27 K.

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