Tissue engineering therapies require biocompatible and bioactive biomaterials that are capable of encouraging an angiogenic response for effective tissue regeneration. In this study, a SVVYGLR peptide, which functions as a potent angiogenic factor, was conjugated into injectable gelatin-poly(ethylene glycol)-tyramine (GPT) hydrogels in situ to enhance endothelial cell activities and neo-vascularization. SVVYGLRGGY (SV-Y) conjugated GPT (SV-GPT) hydrogels were formed in situ via enzyme-mediated reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). The physico-chemical properties were characterized and could be controlled depending on the feed peptide and H(2)O(2) concentration. The concentration of conjugated peptide ranged from 0.37 to 0.81 μmol/mL, and the elastic moduli (G') of the hydrogels were 600-4900 Pa. In vitro cell studies using human umbilical vein endothelial cells (HUVECs) and in vivo subcutaneous injection studies were performed to confirm the effect of the SVVYGLR peptide on HUVEC activity and neo-vascularization. Obtained results demonstrated that the in situ conjugation of SVVYGLR sequences into phenol residues of GPT hydrogels enhanced the activity of HUVECs in vitro and stimulated the formation of new blood vessels in the hydrogel matrices in vivo. From the results, we suggest that in situ conjugation of SV-Y to GPT hydrogels via the enzymatic reaction may be an efficient tool to prepare injectable bioactive hydrogels that can enhance endothelial cell activities and promoting angiogenesis for tissue regeneration.