AC electrokinetics is becoming a strategic tool for lab-on-a-chip systems due to its versatility and its high level of integration. The ability to foreseen the behaviour of fluids and particles under non-uniform AC electric fields is important to allow new generations of devices. Though most of studies predicted motion of particles in co-planar electrodes configurations, we explore a pure 3-D AC electrokinetic effect that can open the way to enhance contact-less handling throughout the microchannel. By fabricating 3D microfluidic chips with a bi-layer electrodes configuration where electrodes are patterned on both sides of the microfluidic channel, we present a detailed study of the AC electrokinetic regimes that govern particles motion suspended in different host media subjected to a non-uniform AC electric field that spreads through the cross-section of the microchannel. We simulate and observe the motion of 1, 5, and 10 μm polystyrene particles relative to the electrodes and provide an insight on the competition between electro-hydrodynamical forces and dielectrophoresis. We demonstrate that using relevant electrode designs combined with the appropriate applied AC potential, particles can be handled in 3-D in the micro-channel at a single or a collective level in several medium conductivities. Both numerical simulations and experimental results provide a useful basis for future biological applications.