In the present study, we separated the marker particles from the suspending particle mixture solution using isotacho-electrophoresis technique, a novel quantitative ionic particle separation method, in the microchannel. A multiple stacking zone of the suspending particle was visualized with variations in electric field strength, pH value and concentration of the ionic solution. In particular, the electrophoretic mobility of ionic particle (fluorescein) was estimated based on the electrophoretic velocity value measured by the particle image velocimetry. As a result, isotacho-electrophoresis zones were clearly visualized as going downstream in the electric field. The particle migration velocity increased proportional to the applied voltage increase; it was also affected by the pH value variations in the ionic solution.