Abstract: Collagen is an important part of the extracellular matrix and a suitable scaffold material due to its biocompatibility and low immunogenicity. However, pure collagen alone does not produce satisfactory experimental results. Therefore, it is crucial to optimize methods to construct collagen-based scaffold materials which are better suited to specific structures and properties. In this study, three collagen-based scaffolds were created using different crosslinking methods: 1-ethyl-(3-dimethylaminopropyl) carbamyl diimide (EDC) and N-hydroxy-succinimide (NHS) crosslinking, methacrylate anhydride (MA) modified photopolymerization crosslinking, and phenyl-2,4,6-trimethyl benzoyl lithium phosphate (LAP) photo crosslinking. The microstructure, physical and chemical properties, and biological properties of the scaffolds were evaluated. Results showed that the three scaffolds had different pore structures with pore sizes ranging from 50 μm to 250 μm because the crosslinking processes regulated the arrangement of collagen fibers and the distribution orientation of porous structures. Additionally, the three scaffolds had good moisture retention properties, and Col-LAP scaffolds had the highest water absorption rate of (1521.66±148.43)%. The mechanical properties of the Col-MA scaffolds were significantly improved and met the basic requirements of mechanical properties for ideal scaffolds for tissue engineering. Being compressed to 50%, the Col-MA scaffolds achieved a compressive strength of 2.17 MPa. The results of cytotoxicity test and fluorescence staining showed that the three scaffolds had good biocompatibility. In conclusion, the differences in microstructure and physical and chemical properties of scaffolds prepared by the three cross-linking methods can provide new references and ideas for future screening of tissue engineering scaffolds.