Cartilage engineered from chondrocytes requires a scaffold to keep the cells in the cartilage defect and to act as a support for inducing hyaline cartilage formation on occasion. In this study, we developed a novel three-dimensional special scaffold in combination with a controlled release of bFGF, which provided structural support and stimulated repair. Gelatin microspheres loaded with bFGF (GM-bFGF) showed a fast release at the initial phase (28.23%) and the ultimate accumulated release was 92.9% by day 14. Three-dimensional gelatin-chondroitin-hyaluronan hybrid scaffolds seeded with cultured autologous chondrocytes were transplanted into the defects in rabbit knees and analyzed histologically at 12 and 24 weeks after the operation. Our findings showed that the defects were filled with smooth, shiny white cartilaginous tissue macroscopically and hyaline-like cartilage histologically 24 weeks postoperatively. The present study implied the great potential of the novel scaffold with GM-bFGF as a new way to promote the retention of chondrocytes and it might serve as a desirable cartilaginous tissue scaffolds to enhance the chondrogenesis.