The invention relates to a double-layered nanostructure for regulating circular dichroism and a preparation method thereof. The double-layered nanostructure is a double-layered chiral structure comprising a plurality of gold nano units with the same structure being combined up and down, left and right, and each gold nano unit comprises an upper structure and a lower structure, the lower structurecomprises a lower bar, the upper structure comprises a metal rod and an upper bar located on the side of the metal bar. The angle between the metal bar and the upper bar is 90 degree, the metal bar and the lower bar are placed in parallel, and one end of the lower rod is perpendicular to one end of the upper rod near the metal rod in different surfaces; the preparation method of the double-layerednanostructure comprises the steps of preparing a substrate, coating photoresist, drying after coating, exposing a structure pattern through an electron beam, developing, photographic fixing, drying after photographic fixing, gold plating, stripping PMMA photoresist, blow-drying, plating SiO2 intermediate layer and the like. The circular dichroism produced by the double-layered nanostructure of the invention has a better effect and can also dynamically adjust a circular dichroism signal of the structure.