The preparation and characterization of mono- and multilayers of cellulose acetate (CA) Langmuir-Blodgett films on indium tin oxide and gold surfaces were studied in detail for the first time. These layers were characterized by their thickness, wettability, morphology and structure using various surface techniques. The thickness of a monolayer of CA based on XPS measurement was one nanometre. Multilayers of CA Langmuir films were homogeneously transferred onto solid surfaces. The permeation of different molecules across these films was studied using electrochemistry in various redox solutions. Our findings suggest that a membrane like structure is formed, which is less permeable as the number of layers increases. Finally, potential applications of these ultrathin films as supports for accommodating biomolecules or metal nanoparticles are presented.