Objective To study the relationship between the construction of large engineered bone and flow shear stress inside the β-tricalcium phosphate (β-TCP) scaffold in perfusion bioreactor with the flow model. Method The scaffolds seeded with human bone marrow-derived mesenchymal stem cells were cultured in perfusion bioreactor. The viscosity of the media was 1.12 mPa?s, 223 mPa?s, and 3.35 mPa?s respectively The construction of the tissue-engineered bone was assessed through the proliferation and osteogenic differentiation of cells and histological assay of the constructs. The flow model was established to calculate the flow shear stress inside the β-TCP scaffold. Result The cell viabilitywas highest in the group with the viscosity of 223 mPa?s. The alkaline phosphatase activity at day 28 and the osteocalcin secretion after 7 days were higher in the two groups with the viscosities of 223 mPa?s and 3.35 mPa?s than in the group with the viscosity of 1.12 mPa?s. Increasing the viscosity of the medium resulted in an earlier emergence of secretion peak of osteopontin. After 28 days of culture, the mineralized extracellular matrix formed inside the scaffold was most in the group with the viscosity of 3.35 mPa?s. The averaged flow shear stress in the scaffold was 5 mPa, 11 mPa, and 15 Pa accordingly when the viscosity of the media was 1.12 mPa?s, 223 mPa?s, and 335 mPa?s respectiwly. Conclusions In this study, a 15 mPa of flow shear stress was optimal in the construction of a large-scale tissue-engineered bone using the β-TCP scaffold combined with human bone marrow-derived mesenchymal stem cells in a perfusion bioreactor.