Cartilage lesions are commonly age-related disorders capable of affecting regular joint movement until they extend to underlying subchondral bones, resulting in osteochondral defects. Gradient scaffold as an effective solution for healing has drawn more attention, however, current studies only consider the biomimetic design of gradient structures, but ignore the chiral microenvironment remodeling in native tissues. Here, highly biomimetic chiral gradient scaffolds are designed and prepared by using lyophilization bonding among layers for liquid chitosan (CS) substrate incorporating stratified-distributed chiral hydroxyapatite (HAP). These biomimetic scaffolds facilitate both bone marrow stromal cells (BMSCs) adhesion and differentiation and chondrocytes dedifferentiation inhibition in vitro tests. Furthermore, in vivo assays unveil the presence of the levorotatory (L) in them markedly strengthened the performance of osteochondral regeneration, in stark contrast to the dextrorotatory (D). The subsequent investigation into the repair-promoting mechanism prove L enhanced nuclear transport of yes-associated protein (YAP) by modulating stress fiber distribution in BMSCs, which is instrumental in up-regulation of osteogenesis-related biomarkers. Meanwhile, detected up-regulation of chondrogenesis biomarkers suggest enhanced bone repair provided structural support for neochondrogenesis and averted collapse. These results indicate novel bioactive scaffolds are beneficial for achieving effective repair in the context of osteochondral injury situation.