Here we present a new process to prepare cell co-culture platforms incorporating thin extracellular matrix (ECM) membranes. We utilized PDMS microstencil plates to generate thin ECM membranes, which were physically stabilized in the micro through holes of the stencil plate. An aqueous solution of ECM (type I collagen or Matrigel) was introduced into the micro-holes of the stencil plate, and then it was dried to form a condensed ECM membrane. The thickness of the ECM membrane was controlled from ~0.1 to ~0.5 μm simply by changing the initial concentration of the ECM solution and/or the depth of the micro-holes. We performed heterotypic cell co-culture on both surfaces of the ECM membranes, demonstrating the high usability for mammalian cell culture. The presented thin ECM membranes would be applicable to the reconstruction of cell-friendly environment mimicking the in vivo tissue structures and to the cell-based drug assay systems.