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 in vivo 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 in situ 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 μm), high permeability (<9 × 10−5 cm 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 in vitro 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 in vitro barrier model.