The design and fabrication of a poly(dimethyl-siloxane) (PDMS)-based microfluidic device for microsphere single-pass applications are presented. The fabrication process includes the use of high-throughput hot embossing lithography (HEL) technology for the definition of the microchannels and standard cast molding process. The sealing of the Pyrex cover and the PDMS substrate is achieved using thermal bonding. The chip, including 20-μm microchannels for microsphere single-passing is already realized, and the fact that the size of the microchannels can be altered makes it convenient to be further optimized. Such single-pass separation of microspheres is very useful for the detection and counting of microspheres. As a new method, the design and fabrication of a microcarrier-controlled microfluidic device described here can be applied to flow cytometric systems for automated, integrated, and ultrafast bioassays and screenings.