For the development of a measurement technique for near-wall liquid temperature, the present study proposes two methods based on total internal reflection fluorescence microscopy (TIRFM). One is based on two-color laser induced fluorescence (LIF) which exploits the temperature dependence of the intensity ratio of two fluorescence bands from a single fluorescent dye. The other is based on fluorescence polarization (FP) which exploits the depolarization phenomenon due to the rotational Brownian motion of fluorescent molecules. In these methods, evanescent wave was generated at the interface between glass wall and solution in a microchannel, so that only the fluorescent molecules near the wall within ~100 nm were excited. In two-color LIF method using sulforhodamine B (SrB), the calibration result showed that the fluorescence intensity ratio of 580−590 nm to 612−644 nm decreased with temperature in the range of 25−40 °C. However, the intensity ratio also decreased with time, which is presumably caused by the adsorption of SrB to the glass wall. In FP method using uranine, polarization degree decreased with temperature in the range of 25−40 °C with the sensitivity of 0.7%/°C, which agrees with the theoretical value qualitatively. Temperature distribution measurement was also conducted using FP method in the vicinity of the channel bottom wall on which an Au line heater of 200 nm thick and 20 µm wide was patterned. The measured and calculated temperature distributions of the near-wall liquid agreed well within 1°C in the area more than 50 µm away from the heater in the spanwise direction.