Recently, three-dimensional (3D) bioprinting technology is becoming an appealing approach for osteochondral repair. However, it is challenging to develop a bilayered scaffold with anisotropic structural properties to mimic a native osteochondral tissue. Herein, we developed a bioink consisting of decellularized extracellular matrix and silk fibroin to print the bilayered scaffold. The bilayered scaffold mimics the natural osteochondral tissue by controlling the composition, mechanical properties, and growth factor release in each layer of the scaffold. The in vitro results show that each layer of scaffolds had a suitable mechanical strength and degradation rate. Furthermore, the scaffolds encapsulating transforming growth factor-beta (TGF-β) and bone morphogenetic protein-2 (BMP-2) can act as a controlled release system and promote directed differentiation of bone marrow-derived mesenchymal stem cells. Furthermore, the in vivo experiments suggested that the scaffolds loaded with growth factors promoted osteochondral regeneration in the rabbit knee joint model. Consequently, the biomimetic bilayered scaffold loaded with TGF-β and BMP-2 would be a promising strategy for osteochondral repair.