Currently, a tissue-engineered bone is usually constructed using a perfusion bioreactor in vitro. In the perfusion culture, fluid flow can exert shear stress on the cells seeded on scaffold, improving the mass transport of the cells. This experiment studied the effects of flow shear stress and mass transport, respectively, on the construction of a large-scale tissue-engineered bone using the critical-sized beta-tricalcium phosphate scaffold seeded with human bone marrow-derived mesenchymal stem cells (hBMSCs). This was done by changing flow rate and adding dextran into the media, thus changing the media's viscosity. The cells were seeded onto the scaffolds and were cultured in a perfusion bioreactor for up to 28 days with different fluid flow shear stress or different mass transport. When the mass transport was 3 mL/min, the flow shear stress was, respectively, 0.005 Pa (0.004-0.007 Pa), 0.011 Pa (0.009-0.013 Pa), or 0.015 Pa (0.013-0.018 Pa) in different experiment group obtained by simulation and calculation using fluid dynamics. When the flow shear stress was 0.015 Pa (0.013-0.018 Pa), the mass transport was, respectively, 3, 6, or 9 mL/min. After 28 days of culture, the construction of the tissue-engineered bone was assessed by osteogenic differentiation of hBMSCs and histological assay of the constructs. Extracellular matrix (ECM) was distributed throughout the entire scaffold and was mineralized in the perfusion culture after 28 days. Increasing flow shear stress accelerated the osteogenic differentiation of hBMSCs and improved the mineralization of ECM. However, increasing mass transport inhibited the formation of mineralized ECM. So, both flow shear stress and transport affected the construction of the large-scale tissue-engineered bone. Moreover, the large-scale tissue-engineered bone could be better produced in the perfusion bioreactor with 0.015 Pa (0.013-0.018 Pa) of fluid flow shear stress and 3 mL/min of mass transport.