We have developed a fluorescence-based microfluidic system for high-throughput analyses of the function of ATP-binding cassette (ABC) transporters. Our device consists of polydimethylsiloxane (PDMS) and a glass that tethers vesicles on its surface. Although PDMS is a great material for fabrication of microfluidic channels, it absorbs fluorescent molecules and hampers quantitive fluorescence analyses. We found that conformal deposition of parylenes on the inner surface of PDMS channels dramatically suppressed the absorption of a fluorescent dye, Rhodamine B. In the parylene-coated PDMS channels, immobilized ABC-transporter (MDR1) vesicles exhibited an ATP-dependent transport of a fluorescent substrate, Rhodamine 123.