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In this study, inspired by the components of cartilage matrix, a photo-cross-linked extracellular matrix (ECM) bioink composed of modified proteins and polysaccharides was presented, including gelatin methacrylate, hyaluronic acid methacrylate, and chondroitin sulfate methacrylate. The systematic experiments were performed, including morphology, swelling, degradation, mechanical and rheological tests, printability analysis, biocompatibility and chondrogenic differentiation characterization, and RNA sequencing (RNA-seq). The results indicated that the photo-cross-linked ECM hydrogels possessed suitable degradation rate and excellent mechanical properties, and the three-dimensional (3D) bioprinted ECM scaffolds obtained favorable shape fidelity and improved the basic properties, biological properties, and chondrogenesis of synovium-derived MSCs (SMSCs). The strong stimulation of transforming growth factor-beta 1 (TGF-β1) enhanced the aggregation, proliferation, and differentiation of SMSCs, thereby enhancing chondrogenic ECM deposition. In vivoanimal experiments and gait analysis further confirmed that the ECM scaffold combined with TGF-β1 could effectively promote cartilage regeneration and functional recovery of injured joints. To sum up, the photo-cross-linked ECM bioink for 3D printing of functional cartilage tissue may become an attractive strategy for cartilage regeneration.