To find an optimal approach for preparation of porous scaffold for tissue engineering, natural cancellous bone were processed with several techniques: calcination, lyophilization, treatment of chemical reagents and supercritical CO 2 fluid extraction, respectively. Scanning electron micrograph, energy dispersive X-ray microanalysis, X-ray diffraction, Micro-CT, mechanical test and T-cell proliferation assay were used to observe and compare morphology, microstructure, composition, mechanical strength and biocompatibility of the different scaffolds. After processing all the scaffolds exhibited natural porous three-dimension microstructure, and the composition and properties of natural bone, such as Ca/P ratio and hydroxyapatite crystal were still preserved in all of them. Except scaffolds processed with lyophilization, all the other scaffolds showed no influence on the proliferation of T-cell. And scaffolds processed with supercritical CO 2 fluid performed higher compressive strength than that of the other scaffolds, which was similar to natural bones. As the consequence, supercritical CO 2 fluid extraction may be a useful approach for preparation of natural scaffolds for tissue engineering.