The invention belongs to the technical field of micro-nano optics, and particularly relates to a method for preparing a double-layer rectangular hole micro-nano structure generating asymmetric transmission. The method for preparing the double-layer rectangular hole micro-nano structure comprises the steps of configuration designing, substrate preparing, nickel plating, gold plating, medium plating, gold plating and bombardment. According to the method for preparing the double-layer rectangular hole micro-nano structure, all nano layers are firstly evaporated, then rectangular hole etching is precisely carried out through a focused ion beam (FIB), the problem that in the prior art, rectangular holes in the upper layer and the lower layer in a double-layer rectangular hole micro-nano structure are hard to calibrate due to the limit of technical and experimental errors is solved, and the advantages of simplifying the preparation process, reducing the preparation difficulty and reducing the cost are achieved.