In this work, we have designed and fabricated a threedimensional hydrodynamic focusing microfluidic device. The device comprises two-layer PDMS microchannels structure. Sample flow stream was firstly vertically constrained into a narrow core-region, and then horizontally focused into one small window from cross-section perspective, which is useful for cell/particle counting. We have show the numerical and experimental images of the focused stream shape from cross-section perspective, experimental ones were captured using confocal fluorescence microscope. We have also investigated the effect of channel aspect ratio on vertical focusing effect using CFD simulations. The results show that the sample flow can be focused successfully in lower aspect ratio (~0.5) of main channel in our design. Furthermore, the effect of Reynolds number on the vertical focusing effect was also investigated. The numerical results show that the rectangular-like shape of the focused stream from cross-section perspective was deformed as Reynolds number is high due to stronger secondary flows was produced in vertical focusing element. This phenomenon was also demonstrated experimentally. In other words, the chip only works well at low Reynolds number (< 5). The device can be integrated into on-chip flow cytometry.