Objective To evaluate the biocompatibility of MC3T3-E1 cells with novel biomimetic multicomponent nano-hydroxyapatite-based biomaterials and observe the attachment, proliferation and shape of MC3T3-E1 cells on the biomaterial. Methods The novel biomimetic multicomponent nano-hydroxyapatite-based biomaterials were prepared from chitosan, hydroxyapatite, gelatin and pectin in certain ratio by biomimtic approach. MC3T3-E1 cells were seeded on the scaffolds, and the biocompatibility was studied by means of the inverted microscope, HE staining, the scanning electron microscope, methyl thiazolyl tetrazolium(MTT), F-DA and Hoechst33258 staining. Results The novel biomimetic multicomponent nano-hydroxyapatite-based biomaterials were three-dimensional and had more porous. MC3T3-E1 cells were seeded on the scaffolds, the cells attached and grew well in the scaffolds.The polygonal or fusiform MC3T3-E1cells were observed in the scaffold. The results of MTT assay showed that the comparison of absorbance in the scaffold group and control group had no statistical significance. Conclusion The multicomponent nano-hydroxyapatite-based biomaterials have well-developed, morphology, physicochemical properties and superior cytocompatibility. They can be potential candidate materials for bone tissue engineering.