Organ-on-a-chip (OoC) is one of the most popular microfluidic chips and possesses various industrial, biomedical, and pharmaceutical applications. So far, many types of OoCs with different applications have been fabricated, most of which contain porous membranes useful as cell culture substrates. One of the challenging parts of OoC’s chips is porous membrane fabrication, making it a complex and sensitive process, which is an issue in microfluidic design. These membranes are made of various materials, the same as biocompatible polymer polydimethylsiloxane (PDMS). Besides OoC, these PDMS membranes can be applied in diagnosis, cell separating, trapping, and sorting. In the present study, a new approach has been presented to design and fabricate an efficient porous membrane in terms of time and cost. The fabrication method has fewer steps than previous techniques and employs more conventional approaches. The presented method for membrane fabrication is functional and a novel way to continue producing this product with a single mold and peeling off the membrane on each try. Merely one sacrificial layer (polyvinyl alcohol) and an O2 plasma surface treatment have been used for fabrication. Surface modification and sacrificial layer on the mold ease the peeling of the PDMS membrane. Transferring process of the membrane to the OoC device is explained, and a filtration test is presented to show the functionality of the PDMS membranes. Cell viability is investigated by MTT assay to ensure the PDMS porous membranes are suitable for microfluidic devices. Also, cell adhesion, cell count, and confluency are analyzed, showing almost the same results for the PDMS membranes and the control samples.