This study presents a novel 3-D hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is compressed initially in the horizontal direction by a set of sheath flows such that it is constrained to the central region of the microchannel and is then focused in the vertical direction by a second pair of sheath flows. Thereafter, the focused sample stream passes over a micro-weir structure positioned directly beneath an optical detection system to capture polystyrene beads fluorescent signal. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations. An experimental investigation is then performed using a micro-flow cytometer fabricated using conventional micro-electro-mechanical systems techniques and an isotropic wet etching method. The results indicate that the two sets of sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure prompts the separation of a mixed sample of 5 and 10 microm polystyrene beads in the vertical direction and ensures that the beads flow through the detection region of the microchannel in a sequential fashion and can therefore be reliably detected and counted.