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A porous chitosan-gelatin scaffold with specific geometry and complex internal microarchitectures was fabricated via a novel indirect rapid prototyping technique. A stereolithography (SL) technique was employed to build the resin mold, based on which a high-resolution poly-dimethylsilicone (PDMS) mold was microreplicated. The chitosan-gelatin hybrid solution was cast on the PDMS mold for pre-freezing and the monolayer scaffold with predefined features and highly porous structures was produced upon lyophilization. These monolayer structures along with the special interface layers can be stacked together to form a three-dimensional (3D) scaffold. The morphologies of the predefined features maintain well during the assembly process. Hepatocyte culture experiment indicates that the novel microstructured chitosan-gelatin porous scaffold could facilitate cells to migrate and proliferate into the deep layers of the scaffold in comparison with the traditional porous only scaffold. This approach provides a new way to fabricate a complex 3D scaffold using various natural biomaterials for vital organ engineering. [ABSTRACT FROM AUTHOR]