Objective To construct the three-dimensional (3D) fluid model at the physiological level of shear stresses and study the effects of fluid shear stress (FSS) on adhesion, differentiation and mechanical sensitivity of osteoblasts. Methods The MC3T3-E1 osteoblasts cultured on β-tricalcium phosphate (β-TCP) scaffolds were subjected to various FSSs in the perfusion flow chamber for 6 hours to compare cell adhesion in FSS-loading groups and control group. Nitric oxide (NO) and alkaline phosphatase (ALP) were detected to compare mechanical sensitivity and cell differentiation. The FSS magnitude and distributions corresponding to various fluid rates were calculated with nonlinear fluid-structure coupling analysis. Results Cell adhesion rate was up to 74%~81% when the average FSS magnitude was lower than 0.4 Pa, but reduced to 60.22% when the average FSS was 0.41 Pa. The NO production rate reached the maximal concentration after loading for 5 min, then significantly reduced at 15 min, and gradually diminished to none at 30 min. ALP level significantly increased (P<0.01) at the shear stress range of 0.232 ~ 0.304 Pa, but maintained at the range of 0.304 ~ 0.412 Pa (P>0.05) with the increase of shear stress. Conclusions Majority of the cells kept a normal adherence to the scaffold at the physiological level of shear stresses. The mechanical sensitivity of the cells under 3D condition was dependent on the FSS rate, which was consistent with two-dimensional (2D) condition. When the average FSS was lower than 0.304 Pa in the scaffold, FSS could significantly promote cell differentiation, but no significant change in cell differentiation could be found when FSS was higher than 0.304 Pa. The present study is expected to accelerate the realization of bone tissue engineering.