A　dual-layer　biomimetic　cartilage　scaffold　was　prepared　by　mimicking　the　structural　design,　chemical　cues　and　mechanical　characteristics　of　mature　articular　cartilage.　The　surface　layer　was　made　from　collagen　(COL),　chitosan　(CS)　and　hyaluronic　acid　sodium　(HAS).　The　transitional　layer　with　microtubule　array　structure　was　prepared　with　COL,　CS　and　silk　fibroin　(SF).　The　PLAG　microspheres　containing　kartogenin　(KGN)　and　the　polylysine-heparin　sodium　nanoparticles　containing　TGF-β1　(TPHNs)　were　constructed　for　the　surface,　transitional　layer,　respectively.　The　SEM　result　showed　that　the　dual-layer　composite　scaffold　had　a　double　structure　similar　to　natural　cartilage.　The　vitro　biocompatibility　experiment　showed　that　the　biomimetic　cartilage　scaffold　with　orientated　porous　structure　was　more　conducive　to　the　proliferation　and　adhesion　of　BMSCs.　A　rabbit　KOA　cartilage　defect　model　was　established　and　biomimetic　cartilage　scaffolds　were　implanted　in　the　defect　area.　Compared　with　the　surface　layer　and　transitional　layer　scaffolds　group,　the　results　of　dual-layer　biomimetic　cartilage　scaffold　group　showed　that　the　defects　had　been　completely　filled,　the　boundary　between　new　cartilage　and　surrounding　tissue　was　difficult　to　identify,　and　the　morphology　of　cells　in　repair　tissue　was　almost　in　accordance　with　the　normal　cartilage　after　16　weeks.　All　those　results　indicated　that　the　biomimetic　cartilage　scaffold　could　effectively　repair　the　defect　of　KOA,　which　is　related　to　the　fact　that　the　scaffold　could　guide　the　morphology,　orientation,　and　proliferation　and　differentiation　of　BMSCs.　This　work　could　potentially　lead　to　the　development　of　multilayer　scaffolds　mimicking　the　zonal　organization　of　articular　cartilage.　©　2019,　©　2019　The　Author(s).　Published　by　Informa　UK　Limited,　trading　as　Taylor　&　Francis　Group.