Objective Prepare biomimetic multilayered scaffold which has similar structure of natural cartilage. Methods By lyophilizing the scaffolds which were prefrozen at -20℃ and in liquid nitrogen successively, we prepared double-layered spongy scaffolds. By partially thawing the prefrozen samples and refreezing them in liquid nitrogen before the final liyophilization, we prepared biomimetic multilayered scaffolds. XRD and FT-IR were used to confirm the interaction between collagen and chitosan. SEM was used to observe the morphologies of the scaffolds. The mechanical properties of pure chitosan scaffolds, pure collagen scaffolds, composite single-layered scaffolds and biomimetic multilayered scaffolds were compared. Results There is chemical interaction between collagen and chitosan. Composite materials eill form better pore structure. The biomimetic multilayered scaffolds had upright pores, round pores and a dense layer from bottom to top of the scaffolds. The scaffolds had quite different mechanical properties between dry and wet state. Under wet state, the different layers of the biomimetic multilayered scaffolds have different mechanical properties. Conclusions The biomimetic structure of the multilayered scaffold is very close to that of the natural articular cartilage, and the different layers of the biomimetic multilayered scaffolds had different mechanical properties under wet state. These are hopefully beneficial to help maintain the phenotypes of chondrocytes and promote the repairing effect of cartilage defects .