An extracellular matrix (ECM) membrane made up of ECM hydrogels has great potentials to develop a physiologically relevant organ-on-a-chip because of its biochemical and biophysical similarity to<i>in vivo</i>basement membranes (BMs). However, the limited mechanical stability of the ECM hydrogels makes it difficult to utilize the ECM membrane in long-term and dynamic cell/tissue cultures. This study proposes a thin but robust and transparent ECM membrane reinforced with silk fibroin (SF)/polycaprolactone (PCL) nanofibers, which is achieved by<i>in situ</i>self-assembly throughout a freestanding SF/PCL nanofiber scaffold. The SF/PCL nanofiber-reinforced ECM (NaRE) membrane shows biophysical characteristics reminiscent of native BMs, including small thickness (<5<i>μ</i>m), high permeability (<9 × 10^-5cm s^-1), and nanofibrillar architecture (∼10-100 nm). With the BM-like characteristics, the nanofiber reinforcement ensured that the NaRE membrane stably supported the construction of various types of<i>in vitro</i>barrier models, from epithelial or endothelial barrier models to complex co-culture models, even over two weeks of cell culture periods. Furthermore, the stretchability of the NaRE membrane allowed emulating the native organ-like cyclic stretching motions (10%-15%) and was demonstrated to manipulate the cell and tissue-level functions of the<i>in vitro</i>barrier model.