Biomimetic three-layered monolithic scaffold (TLS) intended for treatment of osteochondral defects was fabricated by using freeze drying method. The multilayered material was prepared with chitosan (C) and alginate (A) polyelectrolyte complex (CA/PEC) as a cartilaginous layer, a combination of CA/PEC (60 wt%) and beta-tricalcium phosphate (beta-TCP) (40 wt%) as an intermediate layer and a combination of CA/PEC (30 wt%) and beta-TCP (70 wt%) as a subchondral layer in order to mimic the inherent gradient structure of healthy osteochondral tissue. Characterization of the scaffolds was performed using Fourier transform infrared (FT-IR) spectroscopy analysis, swelling and scanning electron microscopy (SEM) tests. In vitro cytotoxicity assay with L929 cells and EpiDerm skin irritation test (SIT) using the EpiDerm reconstructed human epidermal (RHE) model were performed to analyze biocompatibility of the scaffolds. Characterization results showed that there were strong ionic interactions among chitosan, alginate and beta-TCP and the layers showed interconnected porous structure with different swelling ratios. The relative cell viability and SIT results were greater than 70% indicating that the scaffolds are considered nontoxic according to the International Organization for Standardization (ISO) standard. All results taken together, biomimetic TLS can be considered to be suitable for osteochondral applications. (C) 2015 Elsevier B.V. All rights reserved.