The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen-nanohydroxyapatite beads. We also investigate the effect of the collagen-nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen-nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen-nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.