The invention discloses a multi-layer metamaterial unit structure and a preparation and regulation method of the multi-layer metamaterial unit structure. The multi-layer metamaterial unit structure comprises surface layer metal, an upper layer medium, middle layer metal, a lower layer medium and bottom layer metal sequentially from top to bottom, wherein the surface layer metal and the middle layer metal are composed of metal rings of different sizes, an overlapped area and a non-overlapped area exist between the two layers of metal rings at the same time, and the bottom layer metal of the structure is a continuous metal film. The performance of the multi-layer metamaterial can be regulated through the traditional methods of changing resonance metal graphs, metal and medium material parameters and the like, more importantly, and the response frequency and the frequency band of the metamaterial relative to incident electromagnetic waves are regulated by changing the diameter of the surface layer metal ring and the middle layer metal ring, the width of the overlapped area of the two rings and the width of the non-overlapped area of the two rings and the like. Compared with a traditional metamaterial structure, the multi-layer metamaterial unit structure has the advantages of having a wider response frequency band, more centralized medium loss, thinner element materials and the like.