Electric field flow fractionation (EFFF) is a powerful separation technique based on an electrical field perpendicular to a pressure-driven flow. Previous studies of microelectric field flow fractionation (μ-EFFF) indicate that separation performance was limited due to a weak effective electric field caused by polarization layers on the electrode surfaces. In this work, we report on a μ-EFFF device that uses a pulsed voltage scheme to overcome these limitations. The device was fabricated in indium tin oxide (ITO)-coated glass with ITO as electrodes. The effective electric field for pulsed voltage operation was found to be 50-fold stronger when compared with constant voltage operation. A strong influence of pulsed voltage frequency on nanoparticle retention times was observed. Using pulsed voltage, improved separation of polystyrene particles of different surface charge and particle size is demonstrated. Pulsed voltage also offers more parameters compared to the constant voltage mode, e.g., pulse frequency, duty cycle, and waveform to optimize the retention behavior of analytes.