Abstract: Objective To establish a collagen hydrogel three-dimensional culture model with rat calvarial osteoblasts (ROBs). Methods ROBs were obtained through enzyme digestion of segregated neonatal SD rat skull. The collagen hydrogel three-dimensional culture model was established by mixing ROBs with different concentrations of type Ⅰ rat tail collagen (collagen concentration of 1, 2, 3 mg/mL), DMEM medium and NaOH under adjusted PH and a temperature of 37 ℃. Cell viability and activity were detected by FDA/PI staining and CCK-8 3 d after cell culture. The optimal culture method of 3D collagen hydrogel was identified. Cell distribution was observed using scanning electron microscopy and HE staining. Results ROBs collagen was formed firmly at 2 mg/mL, which had significantly higher levels of cell viability and activity than those at 1 mg/mL and 3 mg/mL. Scanning electron microscopy and HE staining showed that cells under the 2 mg/mL collagen culture system adhered with collagen tightly and distributed homogeneously. Conclusion A collagen hydrogel 3D culture model was established successfully by mixing ROBs with collagen at 2 mg/mL.