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This invention relates to a three-dimensional scaffold based on chirality and peptides for selective cell adhesion, its preparation method, and its applications, relating to the field of medical products. The three-dimensional scaffold is a stacked, three-dimensional ordered structure, with each layer comprising several ordered polyelectrolyte multilayer membrane soft carriers arranged at an angle between adjacent layers. Each layer of the polyelectrolyte multilayer membrane is formed by alternating deposition of positively charged chiral molecules grafted with specific recognition groups and negatively charged supramolecular polyelectrolytes on the soft carriers, followed by modification with peptides. The two peptides in adjacent polyelectrolyte multilayer membranes are different and are used to promote the specific adhesion of different cells. The three-dimensional scaffold provided by this invention has specific adhesion properties, thereby avoiding excessive cell proliferation in incorrect locations and helping to prevent problems such as re-injury and stenosis of artificial blood vessels.