Functional artificial livers (FALs), with embedded hepatocytes that perform the functions of a normal liver, have been developed during the past decades. It is important to note that the liver scaffold, which is a biologically functional core of bioartificial livers, plays a vital role in the bio-cartridge within a bioartificial liver. In this study, a three-dimensional (3D) liver scaffold for in vitro cultures was fabricated by freeze-drying a chitosan/gelatin (CG) solution. A CG scaffold has advantages such as (i) inexpensive and easy-to-make; (ii) easy to fabricate with varying compressive modulus by changing the concentration of glutaraldehyde; (iii) non-cytotoxicity; and (iv) porous structure is similar to extracellular matrix (ECM), thus facilitating hepatocyte adhesion and proliferation. The results revealed that the compressive modulus and maintainability of a CG scaffold was correlated to the increase in glutaraldehyde. Furthermore, hepatocyte viability and hepatic functions showed the best performances with a 0.61% glutaraldehyde-CG scaffold. This CG scaffold not only had higher hepatocyte biocompatibility and mechanical strength, but also maintained hepatic functions and viability in vitro cultures; especially, the mechanical properties of 0.61% glutaraldehyde-CG scaffold were very similar to those in normal liver. The CG scaffold as a liver scaffold may have high potential for further bioartificial liver design in the near future. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.