This study aimed to prepare a novel organic-mineral nanofiber/hydrogel of chitosan-polyethylene oxide (CS-PEO)/nanoclay-alginate (NC-ALG). The effects of NC particles on the mineralization and biocompatibility of the scaffold were investigated. A layer-by-layer scaffold composed of CS-PEO and NC-ALG was prepared. The morphological properties, swelling, biodegradation, and mechanical behaviors of the scaffolds were evaluated. Furthermore, scaffolds were characterized by the Fourier Transform Infrared (FTIR), the Field Emission Scanning Electron Microscope (FE-SEM), and X-Ray Diffraction (XRD) techniques. Bone-like apatite formation ability of the scaffolds was determined by the mineralization test in a simulated body fluid (M-SBF). In addition, the crystalline phase of bone-like apatite precipitates was investigated by XRD analysis. The cell compatibility of the scaffolds was also studied with osteoblastic cell line MC3T3-E1 by MTT assay. Notably, the incorporation of NC particles in CS-PEO/ALG scaffolds is suitable for bone tissue regeneration which enhances bone-like apatite formation. Further, the hemolysis and MTT assays demonstrated that CS-PEO/NC-ALG scaffold was compatible and safe for MC3T3 cells.