At present, no definitive treatment for articular cartilage defects has been perfected. Most of the previous treatments involved multiple drilling and microfracture over defect sites with repair-related substances, which poses a limited therapeutic effect. End-stage therapy includes artificial knee joint replacement. In this study, we prepared a novel decellularized natural cartilage scaffold from porcine articular cartilage by supercritical CO2 extraction technology and three-dimensional (3D) composites made using decellularized porcine cartilage graft (dPCG) as scaffolds, platelet-rich plasma (PRP), thrombin as signals and chondrocytes as cells for the treatment of articular cartilage defects. In this study, in vitro and in vivo cartilage regeneration and the expression of chondrogenic markers were examined. Decellularized cartilage graft (dPCG) was evaluated for the extent of cell and DNA removal. Residual cartilage ECM structure was confirmed to be type II collagen by SDS PAGE and immunostaining. The new 3D composite with dPCG (100 mg and 2 × 106 chondrocytes) scaffold promotes chondrogenic marker expression in vitro. We found that the in vivo 3D composite implanted cartilage defect showed significant regeneration relative to the blank and control implant. Immunohistochemical staining showed increase of expression including Collagen type II and aggrecan in 3D composite both in vitro and in vivo studies. In this study, the bioengineered 3D composite by combining dPCG scaffold, chondrocytes, and PRP facilitated the chondrogenic marker expression in both in vitro and in vivo models with accelerated cartilage regeneration. This might serve the purpose of clinical treatment of large focal articular cartilage defects in humans in the near future.