In situ forming hydrogel have been widely studied as a cell supporting matrix and protein delivery carrier for tissue regeneration medicine. For an effective regeneration of tissue, various growth factors were used to promote new formation of tissue. In many growth factor loading tools into scaffolds, immobilization method is more effective than others because activity of growth factors was maintained within the hydrogels for a long term. Facile immobilization of growth factors in hyaluronic acid (HA) hydrogels using dual enzymes is reported in the paper. The hydrogels were formed by using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and transforming growth factor-?3 (TGF-?3) was covalently conjugated on the hydrogels in situ using tyrosinase (Ty) without any modifications. For the preparation of hydrogels, HA was grafted with poly(ethylene glycol) (PEG), which was modified with a tyrosine. The gelation times of the HA hydrogels were ranging from 415 to 17 s and the storage moduli was dependent on the concentration of H2O2 and Ty (470-1600 Pa). A native TGF-?3 (200 ng/mL) was readily encapsulated in the HA hydrogels and 17% of the TGF-?3 was released over 1 month at the Ty concentration of 0.5 KU/mL, while the release was faster when 0.3 KU/mL of Ty was used for the encapsulation (27%). It can be suggested that the growth factors resident in the hydrogels for a long period of time may lead cells proliferating and differentiating, whereas the growth factors that are initially released from the hydrogels can induce the ingrowth of cells into the matrices. Therefore, the dual enzymatic methods as facile gel forming and loading of various native growth factors or therapeutic proteins could be highly promising for biomedical applications.