The goal of tissue engineering is to repair or regenerate damaged tissue using a combination of cellular biology and materials engineering techniques. One of the challenging problems in tissue engineering is the development of a reproducible three-dimensional (3D) scaffold to support cell migration and infiltration. Although natural polymers, such as dissolved collagen or alginate, are considered ideal for this purpose, their hydrophilic properties have hindered the fabrication of designed 3D scaffold structures. To overcome this problem, we developed a novel system for the cryogenic plotting of 3D scaffolds. Using this technique, we created various 3D collagen scaffolds with designed pore structures that exhibited desired properties. The diameter of the individual collagen strands, which varied from 250 µm to 500 µm, was reproducibly dependent on processing parameters, and the final collagen scaffold showed little shrinkage (less than 12%) relative to the initial design. To evaluate the fabricated scaffold, we adapted the scaffold to regenerate skin tissue. Immunohistochemical analysis demonstrated that co-cultured keratinocytes and fibroblasts completely migrated throughout the 3D collagen scaffold and keratinocytes were well differentiated on the surface of scaffold like a human skin.