Free-standing lipid bilayers in nano- and micro-pores are interesting membrane models and attractive for biotechnological applications. We describe here the controlled preparation of proteo-lipid mono- and bilayers using the Langmuir-Schaefer transfer or Langmuir-Blodgett technique, respectively on hydrophobic and hydrophilic surfaces. We demonstrate the formation of suspended proteo-lipid layers by Transmission Electron Microscopy (TEM) and in situ Atomic Force Microscopy (AFM) imaging. Using Annexin-A5 as a membrane-associated protein, continuous proteo-lipid mono- and bilayers were formed, which span pore arrays over areas of several square-micrometers. The 2D organization of proteins associated to lipid monolayer is well preserved during the transfer process and the protein association is Ca(2+)-dependent and therefore reversible. The simple formation and reliable transfer of stabilized free-standing lipid films is a first crucial step to create biomimetic membranes for biotechnological applications and membrane protein research.