Cartilage defects can be difficult to heal, potentially leading to complications such as osteoarthritis. Recently, a tissue engineering approach that uses scaffolds and growth factors has been proposed to regenerate new cartilage tissues. Herein, we investigated the application of hyaluronic acid (HA) gel loaded with transforming growth factor-beta 3 (TGF-β3) for enhanced cartilage regeneration. We assessed the clinical conditions required to efficiently enhance the ability of the modified HA gel to repair defective cartilage. Based on our findings, the prepared HA gel exhibited good physicochemical and mechanical properties and was non-toxic and non-inflammatory. Moreover, HA gel-loaded TGF-β3 (HAT) had improved biocompatibility and promoted the synthesis of cartilage-specific matrix and collagen, further improving its ability to repair defects. The application of HAT resulted in an initial burst release of HA, which degraded slowly in vivo. Finally, HAT combined with microfracture-inducing bone marrow stem cells could significantly improve the cartilage microenvironment and regeneration of cartilage defects. Our results indicate that HA is a suitable material for developing growth factor carriers, whereas HAT is a promising candidate for cartilage regeneration. Furthermore, this differentiated strategy provides a rapid and effective clinical approach for next-generation cartilage regeneration.