When two immiscible liquids make contact in a microchannel, the flow pattern is affected by the affinity between channel walls and liquids. In this study, microchannels (200 μm in width and 200 μm in depth) having a Tshaped bifurcation point were fabricated on PMMA plates. The inner walls of the microchannels were modified in a zone-selective manner to be either hydrophilic or hydrophobic, based on verification accomplished via a laser interference fringe technique. The microchannel was placed horizontally, and water and octane were introduced into the upper-side channel (hydrophilic) and into the lower-side channel (hydrophobic), respectively. The experimental results showed that water and octane formed a stable layered flow, and the two liquids were virtually completely separated at the T-shaped section, even when static pressure was intentionally applied to the outlets. CFD simulation, using FLUENT 6.3 software, was performed to explain the role of zone-selective modification of microchannel walls.