Numerous studies about bone matrix fabrication focus on how the species and concentrations of components affect the cellular response. However, there are few studies that investigate how the related spatial arrangement of the components influences cellular activity. The aim of this work was to develop a novel method to biomimetically manufacture a three-dimensional mineral bone matrix and study the effect of apatite-collagen-chondroitin sulfate (CS) microspheres on the adhesion rate and activity of osteoblast-like cells. Although previous studies used a crosslinking agent or lyophilized methods to fabricated three-dimensional collagen microspheres, we produced beads composed of collagen and CS under mild reaction conditions. This process not only maintains collagen self-assembly into fibrils with a D-periodic pattern ability but also simultaneously introduces two major native bone matrix elements, collagen and CS, into the beads. Furthermore, we mimic the native in vivo bone matrix formation process by the direct nucleation and growth of apatite crystals on collagen fibrils. The apatite crystals are similar in composition to human bone mineral via X-ray diffraction and energy-dispersive X-ray spectrometric analysis. The cellular attachment rate of MG63 osteoblast-like cells is significantly higher for collagen-CS-apatite gel beads than for collagen-CS gel beads. In addition, with regard to the osteoblast bioactivity, we observed that alkaline phosphatase activity of MG63 cells on the collagen-CS-apatite gel beads higher than on the collagen-CS gel beads on day 14.