Cartilage defects are among the most difficult diseases to cure in clinic. Due to the limited regeneration capacity of chondrocytes, cartilage regeneration is very difficult. Tissue engineering is a potential strategy for cartilage regeneration. The choice of scaffold is a key factor for the successful construction of tissue engineering cartilage. In this research, we successfully constructed the silk/silk fibroin/gelatin/polylactic acid porous microspheres (S/SF/G/PLLA-PMs) scaffold, then further evaluated the physical and chemical properties and biocompatibility of the composite cartilage tissue in vitro and in vivo, also the long-term survival of the composite cartilage in large animals was carried out. The research results showed that S/SF/G/PLLA-PMs composite scaffold had good biocompatibility. The addition of L-polylactic acid porous microspheres (PLLA-PMs) could significantly enhance the mechanical strength of the scaffold and achieve a multi-level pore structure. After 4 weeks of culture in vitro, composite cartilage could be constructed. Further immunohistochemical results showed that S/SF/G/PLLA-PMs scaffold could increase the long-term stability of the composite cartilage transplantation in vivo.