Membrane fabrication and integration with microfluidic devices has received increased attention for applications including bio-detection (a device providing analytical information in a selective and quantitative manner using a biological recognition element), membrane-based separation, and biological sample purification. The main challenges associated with these applications have been: 1) meeting sensitivity/selectivity requirements, 2) decreasing costs, 3) maintaining the mechanical stability of the membrane, 4) offering high throughput. Therefore, the main goal of this study was to demonstrate size-based membrane separation and bio-detection using double layer channel developed in our lab and to show how the membrane integrated channel can selectively separate rod shape cell separation with various aspect ratio based on size and enhance bio detection rate with flow. Based on an existing double-channel and cross-flow microfluidics platform, we explored various polymeric materials for fabricating porous membranes to use in pore-size-dependent separation. We induced pores via stop-flow lithography, and investigated membrane properties and limitations for pore-size-dependent separation. We investigated potential applications of poly(ethylene glycol) diacrylate (PEGDA)-based membrane integrated platforms in biological molecule detection based on streptavidin and biotin interaction. We demonstrated that flow and concentrations can enhance target detection in this platform.