Hydrodynamic focusing technique to generate focused flow has been used for flow cytometry in microfluidic devices. However, devices with circular capillary tubes made of glass are not suitable for flow visualization or optical signal detection because the rays of light are distorted at the curved interface. We devised a new acrylic chamber assembled with a pulled micropipette and a rectangular microchannel made of glass. This new channel geometry enabled us to visualize the three-dimensional (3D) flow characteristics with confocal imaging technique. We analyzed the 3D hydrodynamic focusing in a circular capillary tube and a rectangular microchannel over a practical range of flow rates, viscosities and pressure drops.