Large area chiral supramolecular self-assembly of rubrene has been achieved on Au(111) surface at room temperature. The basic building block of such self-assembled layers consists of two double-layered Y-shape supramolecular structures containing eight twisted rubrene molecules. Chirality is maintained and transferred from the bottom layer to the top layer in the successive molecular layers for up to five layers. Such chiral multilayers can be potential candidates in enantioselective catalysis and chiral separations.