This study explores the use of silk fibroin-DNA (SF-DNA) hydrogel microspheres (RSD-MSs) for cartilage organoid precursor (COP) construction and cartilage repair. RSD-MSs, developed through a microfluidic system integrating photopolymerization and self-assembly techniques, were modified with Pep-RGDFA to enhance cell adhesion and chondrogenic differentiation. In vitro studies demonstrated that RSD-MSs promoted bone marrow mesenchymal stem cell (BMSC) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis revealed that RSD-MSs induced chondrogenesis through integrin-mediated adhesion pathways and glycosaminoglycan (GAG) biosynthesis. In vivo experiments showed that COPs created using RSD-MSs significantly enhanced cartilage regeneration in a rat model. The study concludes that RSD-MSs are an ideal candidate for constructing and long-term culturing of cartilage organoids, offering a novel strategy and material for cartilage regeneration and tissue engineering.This study explores the use of silk fibroin-DNA (SF-DNA) hydrogel microspheres (RSD-MSs) for cartilage organoid precursor (COP) construction and cartilage repair. RSD-MSs, developed through a microfluidic system integrating photopolymerization and self-assembly techniques, were modified with Pep-RGDFA to enhance cell adhesion and chondrogenic differentiation. In vitro studies demonstrated that RSD-MSs promoted bone marrow mesenchymal stem cell (BMSC) proliferation, adhesion, and chondrogenic differentiation. Transcriptomic analysis revealed that RSD-MSs induced chondrogenesis through integrin-mediated adhesion pathways and glycosaminoglycan (GAG) biosynthesis. In vivo experiments showed that COPs created using RSD-MSs significantly enhanced cartilage regeneration in a rat model. The study concludes that RSD-MSs are an ideal candidate for constructing and long-term culturing of cartilage organoids, offering a novel strategy and material for cartilage regeneration and tissue engineering.