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In this study, novel porous cytocompatible scaffolds with a 3D nanocomposite structure were synthesized by using nanoclay particles embedded into a biopolymer blend composed of polyvinyl alcohol (PVA) and chitosan (CS). According to the results, the Fourier transform infrared spectrum confirmed the presence of nanoclay, PVA and CS in the scaffold structure. X-ray diffraction outcomes showed the enhancement of crystalline zone in the synthesized 3D scaffolds by increasing the nanoclay content. Scanning electron microscopy (SEM) images revealed the highly porous interconnected microstructure of the scaffolds. Also, the energy-dispersive X-ray spectra verified the presence of nanoclay, PVA and CS in the sample with the highest nanoclay content. According to mechanical properties and porosity of the synthesized 3D scaffolds, compressive strength (i.e., 3.5 ± 0.2 MPa), elastic modulus (1.42 ± 0.02 GPa) and porosity (75-82%) of the sample with the highest nanoclay content was in the range of mechanical properties and porosity of a natural trabecular bone tissue. The swelling of samples in a phosphate-buffered saline solution was less than the swelling in water. In addition, increasing the content of nanoclay decreases the percentage of swelling. Outcomes of cell culture experiments confirmed that the synthesized 3D scaffolds were not toxic and the cell attachment SEM images showed a sufficient attachment of the cell to the interconnected porous structure of the sample. Results suggest that the synthesized 3D scaffold in this study possesses proper microstructure properties and no cytotoxicity to be replaced with natural bone tissues.