Silk fibroin (SF) has attracted attention as a base biomaterial that could be suitable in many applications because of its shape and structure. Highly functional SF has been developed to promote tissue regeneration with heparin conjugation. However, the hydrophobic three-dimensional structure of SF makes it difficult to bind to high-molecular-weight and hydrophilic compounds such as heparin. In this study, sufficient heparin modification was achieved using tyrosine residues as reaction points to improve cellular response. As it was considered that there was a trade-off between the improvement of water wettability and cell responsiveness induced by heparin modification, influences on the structure, and mechanical properties, the structure and physical properties of the SF conjugated with heparin were extensively evaluated. Results showed that increased amounts of heparin modification raised heparin content and water wettability on film surfaces even though SF formation was not inhibited. In addition, the proliferation of endothelial cells and fibroblasts were enhanced when a surface with sufficient heparin assumed its potential in assisting wound healing. This research emphasizes the importance of material design focusing on the crystal structure inherent in SF in the development of functionalized SF materials.