The present invention relates to a method for preparing a dendritic cell, a dendritic cell prepared thereby and a use thereof, and more specifically, to a method for preparing a dendritic cell, including: treating a dendritic cell at a maturation stage rather than at an immature stage with an antigen bound to a peptide having a cell membrane permeability to prepare a dendritic cell with improved antigen-presenting ability, a dendritic cell prepared by the method, and an immunotherapeutic agent thereof, a use for anti-tumor vaccines, or a pharmaceutical composition for treating tumors, containing the same.