In this paper, a technique of simultaneously measuring the velocity and the temperature in micro-scale flow is proposed. This method uses particle tracking velocimetry (PTV) for measuring the velocity and laser induced fluorescence (LIE) for measuring the temperature. To measure the accurate velocity and temperature, images for PTV and for LIE are separated by using two light sources and a shutter which is synchronized with a camera. By using only one camera, measurement system can be simplified and the error from complicate optical system can be minimized. Error analyses regarding the concentrations of fluorescent dye and particle and the light source fluctuation are also conducted. It is found that the error of the temperature and the velocity highly depends on the concentration of fluorescent particles which are used for PTV. This technique is applied to the simultaneous measurement of the velocity and the temperature in the electrokinetic flow. It is found that the velocity and temperature vary with the electric field strength and the concentration of electrolyte.