Electrokinetic manipulation refers to the control of particle and cell motions using an electric field. It is an efficient technique for microfluidic applications with the ease of operation and integration. It, however, suffers from an intrinsic drawback of low throughput due to the linear dependence of the typically very low fluid permittivity. We demonstrate in this work a significantly enhanced throughput for electrokinetic manipulation of particles and cells by the use of multiple parallel microchannels in a two-layer stacked microfluidic device. The fabrication of this device is simple without the need of a precise alignment of the two layers. The number of layers and the number of microchannels in each layer can thus be further increased for a potentially high throughput electrokinetic particle and cell manipulations.