This paper presents a generalization of the hydrodynamic focusing technique to three-dimensions. Three-dimensional (3-D) hydrodynamic focusing offers the advantages of precision positioning of molecules in both vertical and lateral dimensions and minimizing the interaction of the sample fluid with the surfaces of the channel walls. In an ideal approach, 3-D hydrodynamic focusing could be achieved by completely surrounding the sample flow by a cylindrical sheath flow that constrains the sample flow to the center of the channel in both the lateral and the vertical dimensions. We present here design and simulation, 3-D fabrication, and experimental results from a piecewise approximation to such a cylindrical flow. Two-dimensional (2-D) and 3-D hydrodynamic focusing chips were fabricated using micromolding methods with polydimethylsiloxane (PDMS). Three-dimensional hydrodynamic focusing chips were fabricated using the "membrane sandwich" method. Laser scanning confocal microscopy was used to study the hydrodynamic focusing experiments performed in the 2-D and 3-D chips with Rhodamine 6G solution as the sample fluid and water as the sheath fluid.