Cell-derived decellularized matrices are promising cell culture substrates for tissue engineering and regenerative medicine. However, it is required that cell-derived decellularized matrices are fabricated as desirably-shaped three-dimensional (3D) scaffolds for these applications because they do not retain macrostructures of tissue and organs. To fabricate cell-derived decellularized matrices as 3D scaffolds, 3D template scaffolds have been employed. Recently, 3D-printing techniques have been widely used to construct 3D scaffolds. Thus, 3D-printed scaffolds are available to fabricate cell-derived decellularized matrices as desirably-shaped 3D scaffolds. In this study, cell-derived decellularized matrices were prepared on 3D-printed poly (D, L-lactic acid) (PLA) scaffolds. HT-1080 fibrosarcoma cells were seeded on 3D-printed PLA scaffolds and were cultured to deposit extracellular matrix components beneath the cells. After the culture, the cells were removed from cell-scaffold constructs. Additionally, deposited fibronectin was detected on the surface of 3D-printed PLA scaffolds. These results indicate that cell-derived decellularized matrices were successfully prepared as desirably-shaped 3D scaffolds with the aid of 3D-printed scaffolds. Moreover, the matrices exhibited cell adhesiveness. These results indicate that 3D-printing techniques will aid fabrication of cell-derived decellularized matrices as desirably shaped 3D scaffolds. Improved production of such desirably-shaped 3D scaffolds of cell-derived decellularized matrices will expand the applications in tissue engineering and regenerative medicine.