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Open microfluidics is an emerging field of bio/medical applications that need direct energy/matter exchange between microfluids and environment. This paper presents the design, simulation, fabrication, and test of a microfluidic chip for a water-in-oil (WiO) two-phase flow in closed-open-closed microchannels. The chip, fabricated from PDMS using soft lithography, consists of a flow-focusing structure for WiO droplet generation and a long closed-open-closed channel for droplet flow. A negative pressure applied to the end of the channel is used as the driving force for WiO droplets to flow through the open channel. It is found that the negative pressure that is capable of driving a steady flow for a given flow rate, without overflow and air suction, falls into a pressure range instead of being an exact value. The mechanism for the pressure range is investigated theoretically and experimentally and is attributed to the surface tension. Yeast cells have been incorporated in the droplets, and the successful flow through the open channels verifies the function of the chips.