Biocompatible and biodegradable gelatin is a good candidate bioink for use in 3D bioprinting technologies, but viscous gelatin solution has a low printability. In order to improve the poor printability of gelatin, we optimized the rheological properties of gelatin solution. 3D gelatin scaffolds were then cross-linked using physical or chemical methods to maintain the 3D structure. The physicochemical and biological differences between the two types of cross-linked gelatin scaffolds were studied. Scanning electron microscopy images revealed that the morphologies of the resulting cross-linked 3D scaffolds maintained their structural stabilities. The physically cross-linked 3D scaffolds maintained their surface sizes without a significant decrease (less than a 3% reduction in the surface size was observed) after cross-linking. To evaluate the differences in cell affinity by two types of cross-linking method, human dermal fibroblasts cultured on the cross-linked 3D scaffolds. After 14 days of culturing, DNA assays showed that the cell proliferation rate of the physically cross-linked 3D scaffold was 44% higher than that of the chemically cross-linked 3D scaffold. In conclusion, the optimized physically cross-linked 3D scaffold retained its surface size without significant decreases after cross-linking, as required by 3D-printed patient-specific tissue engineered customized scaffolds, despite the use of water-soluble gelatin hydrogels.