Tissue engineering approach offers alternative strategies to develop multi-layered/multi-component osteochondral mimetic constructs to meet the requirements of the heterogeneous and layered structure of native osteochondral tissue. Herein, an iterative overlaying process to fabricate a multi-layered scaffold with a gradient composition and layer specific structure have been developed by combining the natural extracellular matrix (ECM) components-chitosan, type I collagen, type II collagen, nanohydroxyapatite- of the osteochondral tissue in biomimetic compositions. Subchondral bone layer was prepared by using freeze-drying method to obtain 3D porous scaffolds. The calcified cartilage and cartilage layers were prepared by thermal gelation method in the hydrogel form. Osteochondral scaffolds fabricated by iterative overlaying of each distinct layer exhibited a porous, continuous gradient structure and supported cell proliferation in a co-culture of MC3T3-E1 preosteoblasts and ATDC5 chondrocytes. Histology and biochemical analysis showed enhanced extracellular matrix production and demonstrated collagen and glycosaminoglycan deposition. Expression of genes specific for bone, calcified cartilage and cartilage were improved in the osteochondral scaffold. Overall, these findings suggest that iterative overlaying of freeze-dried scaffolds and hydrogel matrices prepared by using ECM components in biomimetic ratios to fabricate gradient, multi-layered structures can be a promising strategy without the need for growth factors. (C) 2020 Elsevier B.V. All rights reserved.