A method for preparing a multi-layer substrate, which includes: forming a first film layer on a substrate, and forming a group of alignment marks in alignment areas of the first film layer; and forming a plurality of subsequent film layers and a top film layer on the first film layer in sequence; in the patterning process for each subsequent film layer, alignment marks in a mask plate for the subsequent film layer are aligned with the alignment marks in the first film layer, and photoresist coated on the subsequent film layer is subjected to exposure; and in a patterning process of the subsequent film layer, photoresist patterns, formed by the alignment marks in the mask plate at pattern positions of the alignment marks of the first film layer when the photoresist coated on the subsequent film layer is subjected to exposure, are removed. The method improves the alignment accuracy between the patterns of the formed subsequent film layer.