This study presents a new active micromixer with high mixing efficiency achieved by means of a gradient distribution of the surface zeta potential controlled by changing the frequency of voltage applied on shielding electrodes. Gradient surface zeta potential is generated by applying a high voltage to inclined buried shielding electrodes. While alternating the frequency of driving voltage, the zeta potential could be changed accordingly, thus providing a significant mixing effect inside microchannels. A theoretical model is proposed to predict the distribution of zeta potential. The results from this model are critically compared with the well-developed three-capacitor model. Additionally, two time-factor scales, the charge time of capacitor and mixing length flow time, are used to predict the optimum frequency. The prediction of optimum frequency, 0.5 Hz, is consistent with experimental results. Moreover, a five-pair inclined shielding electrode with a frequency of 0.5 Hz leads to a significant improvement in the mixing performance of the active micromixer. Numerical results indicate that a localized flow circulation is generated when the control voltage is applied to the inclined shielding electrodes. Furthermore, the streamlines are experimentally observed by using fluorescent beads. The shape of this circulation is dependent on the distribution of gradient zeta potential, which is determined by the arrangement of electrodes. The effects of the number of electrode pairs and the layout of shielding electrodes on the mixing performance of micromixer are also explored both numerically and experimentally. It is revealed that five-pair inclined electrodes at 0.5 Hz provide the highest mixing efficiency. Finally, a reaction between N-benzoyl-L-arginine-p-nitroanilide and trypsin enzyme is performed to verify the capability of micromixers. The experimental results reveal that the reaction can achieve a higher performance indicating a higher mixing efficiency. The active micromixers could be used in microfluidic systems for improving the mixing efficiency and thus enhancing the bioreaction.