Collagen has been extensively investigated as a bioactive material in cartilage tissue engineering. Recombinant humanized collagen type III (rhCol III) possessed excellent biocompatibility and imperative interactions with various cells shows a significant advantage as the starting material of medical devices. To investigate the bioactivation effect of rhCol III in cartilage tissue engineering, methacrylated hyaluronic acid (HA-MA) was prepared and rhCol III was further compounded to establish a photo-crosslinked composite hydrogel (HA-rhCol Ⅲ) platform to study the cartilage regeneration with chondrocytes encapsulated. The results verified that the HA-rhCol III hydrogels could be rapidly formed with stable mechanical properties using the blue light curing system. Meanwhile, the rhCol III could be effectively retained inside the composite hydrogel, which was conducive to maintain its bioactive function for a longer period. In vitro cell experiments confirmed that rhCol III improved the local microenvironment for chondrocytes, which provided abundant adhesion sites and further promoted cell migration, proliferation and differentiation. In vivo results indicated that the composite hydrogels could be conveniently applied to fulfill the cartilage defect in rabbit, and the histological and immunohistological results suggested that cartilage regeneration could be achieved with the application of HA-rhCol Ⅲ composite hydrogels. It could be concluded that the addition of rhCol III could bioactivate the hydrogel and promote the tissue regeneration, showing potential for application in tissue engineering.