Chitosan　(ChS),　Chondroitin　Sulfate　A　(CSA)　and　Hyaluronic　Acid　(HYA)　were　employed　as　organic　matrix,　while　calcium　and　phosphate　salt　solution　were　the　precursors　of　the　inorganic　phase.　The　novel　ChS/CSA/HYA/nHAP　nano-composite　scaffold　with　multilevel　structure　(nano-micron-macro)　were　prepared,　using　the　biomimetic　synthesis　and　freeze-drying　technique.　SEM　shows　that　the　composite　scaffold　possesses　interconnected　porous　structure,　with　the　pore　size　of　45~156　µm　and　porosity　at　(93.78±1.56)%.　The　nano-particles　were　formed　in　situ　with　relatively　homogeneous　distribution　in　the　scaffold.　XRD　and　FTIR　confirm　the　scaffold　in　situ　forms　nano-HAP　crystals.　ChS/CSA/HYA/nHAP　nano-composite　scaffold　has　relative　good　mechanical　properties　with　compressive　strength　of　(0.126±0.073)　MPa　and　elastic　modulus　of　(0.682±0.045)　MPa.　Rat　osteoblast　was　used　to　set　up　in　vitro　evaluation　model.　The　MTT　measurement　shows　the　cytotoxicity　grade　of　the　scaffold　is　0　to　1,　which　indicates　that　the　ChS/CSA/HYA/nHAP　nano-composite　scaffold　has　relative　good　cytocompatibility.　All　the　results　show　that　the　ChS/CSA/　HYA/nHAP　nano-composite　scaffold　may　serve　as　a　good　scaffold　for　bone　tissue　engineering.　©,　2015,　Science　Press.　All　right　reserved.