RNA has emerged as a promising material for nanostructure and microstructure engineering. Although rare, some macroscopic RNA structures have also been constructed using lipid or polymer materials. Here, we report the first example of an enzymatically generated RNA membrane. This robust and free-standing RNA membrane has a macroscopic structure and is generated without any polymer support or complexation. Our RNA membrane is fabricated following two sequential processes, complementary rolling circle transcription and evaporation-induced self-assembly, and its structural and functional properties are rationally controlled by adjusting RNA base pairing. In this study, three types of RNA membranes are fabricated and are used to demonstrate potential applications.