Recently, decellularized extracellular matrix-based bio-ink (dECM bio-ink) derived from animal organs is attracting attention because of its excellent biocompatibility. However, its poor 3D printability and weak mechanical properties remain a challenge. Here, we developed a new dECM bio-ink with enhanced 3D printability and mechanical properties. dECM micro-particles of about 13.4 μ m in size were prepared by decellularizing a porcine liver followed by freeze-milling. The new bio-ink, named as dECM powder-based bio-ink (dECM pBio-ink), was prepared by loading the dECM micro-particles into a gelatin mixture. The usefulness of the dECM pBio-ink was evaluated by assessing its mechanical properties, printability, and cytocompatibility. The results showed that its mechanical properties and 3D printability were greatly improved. Its elastic modulus increased by up to 9.17 times that of the conventional dECM bio-ink. Micro-patterns with living cells were successfully achieved with 93% cell viability. Above all, the new bio-ink showed superior performance in stacking of layers for 3D printing, whereas the conventional bio-ink could not maintain its shape. Finally, we demonstrated that the dECM pBio-ink possessed comparable cytocompatibility with the conventional dECM bio-ink through in vitro tests with endothelial cells and primary mouse hepatocytes.