This study presents a novel approach to enhance cartilage repair using RGD-SF-DNA hydrogel microspheres (RSD-MSs) for cartilage organoid precursor (COP) construction. The RSD-MSs were developed by integrating photopolymerization with self-assembly techniques in a microfluidic system, followed by surface modification with Pep-RGDfKA. The RSD-MSs exhibited uniform size, porous surface, and optimal swelling and degradation properties. In vitro studies showed that RSD-MSs enhanced bone marrow mesenchymal stem cell (BMSC) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis indicated that RSD-MSs induced chondrogenesis through integrin-mediated adhesion pathways and glycosaminoglycan biosynthesis. In vivo studies demonstrated that seeding BMSCs onto RSD-MSs to create COPs significantly enhanced cartilage regeneration. The RSD-MSs provided an ideal candidate for the construction and long-term cultivation of cartilage organoids, offering an innovative strategy and material choice for cartilage regeneration and tissue engineering. The study also highlights the potential of RSD-MSs in promoting chondrogenic differentiation of BMSCs through enhanced cell adhesion and focal adhesion pathways, as well as GAG biosynthesis. In vivo evaluation showed that COPs significantly improved cartilage repair compared to RSD-MSs, with the COP group exhibiting better cartilage regeneration and quality. The RSD-MSs demonstrated favorable biodegradability and biocompatibility, making them suitable for biomedical applications. The study provides valuable insights into the development of cartilage organoids and their potential in regenerative medicine.This study presents a novel approach to enhance cartilage repair using RGD-SF-DNA hydrogel microspheres (RSD-MSs) for cartilage organoid precursor (COP) construction. The RSD-MSs were developed by integrating photopolymerization with self-assembly techniques in a microfluidic system, followed by surface modification with Pep-RGDfKA. The RSD-MSs exhibited uniform size, porous surface, and optimal swelling and degradation properties. In vitro studies showed that RSD-MSs enhanced bone marrow mesenchymal stem cell (BMSC) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis indicated that RSD-MSs induced chondrogenesis through integrin-mediated adhesion pathways and glycosaminoglycan biosynthesis. In vivo studies demonstrated that seeding BMSCs onto RSD-MSs to create COPs significantly enhanced cartilage regeneration. The RSD-MSs provided an ideal candidate for the construction and long-term cultivation of cartilage organoids, offering an innovative strategy and material choice for cartilage regeneration and tissue engineering. The study also highlights the potential of RSD-MSs in promoting chondrogenic differentiation of BMSCs through enhanced cell adhesion and focal adhesion pathways, as well as GAG biosynthesis. In vivo evaluation showed that COPs significantly improved cartilage repair compared to RSD-MSs, with the COP group exhibiting better cartilage regeneration and quality. The RSD-MSs demonstrated favorable biodegradability and biocompatibility, making them suitable for biomedical applications. The study provides valuable insights into the development of cartilage organoids and their potential in regenerative medicine.