This paper presents a detection scheme for analyzing the temperature distribution nearby the channel wall in a microfluidic chip utilizing a temperature-dependent fluorescence dye. An advanced optical microscope system—total internal reflection fluorescence microscope (TIRFM) is used for measuring the temperature distribution on the channel wall at the point of electroosmotic flow in an electrokinetically driven microfluidic chip. In order to meet the short working distance of the objective type TIRFM scheme, microscope cover glass slits are used to fabricate the microfluidic chips. The short fluorescence excitation depth from a TIRFM system makes the intensity information obtained using TIRFM is not sensitive to the channel depth variation which ususally biases the measured results while using a conventional Epi-fluorescence microscope (EPI-FM). Therefore, a TIRFM can precisely describe the temperature profile of the distance within 100 nm of the channel wall where consists of the Stern layer and the diffusion layer for an electrokinetic microfluidic system. Results indicate the proposed TIRFM provides higher measurement sensitivity over the EPI-FM. Significant temperature gradient along the channel depth is experimentally observed. In addition, the measured wall temperature distributions can be the boundary conditions for numerical investigation into the joule heating effect. The proposed method gives a precise temperature profile of microfluidic channels and shows the substantial impact on developing a numerical simulation model for precisely predicting the joule heating effect in microfluidic chips.