Objective To study the effects of oscillatory flow, as well as pore size and porosity of the 3D scaffold on distributions of flow rate and shear stress in perfusion bioreactor, and to propose optimization methods for preparing the 3D decellularized bone scaffold and perfusion bioreactor based on theoretical results. Methods Based on the previously established 3D decelluarized scaffold and perfusion bioreactor for bone tissue engineering in the laboratory, the decelluarized scaffold was simplified as an isotropic porous media. The velocity and shear stress distributions in the bioreactor were further simulated theoretically. Results Under the oscillatory flow, the Darcy shear stress and velocity in the 3D porous scaffold presented a consistently regular pattern. Compared with the unidirectional flow, the difference of velocity and Darcy shear stress decreased at different radius, which could contribute to the homogeneous 3D culture of seed cells in bone tissue engineering. Increasing the inlet perfusion velocity could improve the average Darcy shear stress. Increasing the pore diameter or porosity of the scaffold had no obvious effects on the peak value of velocity, but sharply reduced the average Darcy shear stress. Increasing inlet oscillation frequency could decrease the peak value of velocity and obviously decrease the difference of velocity at different radius. Conclusions Appropriate oscillatory flow was beneficial for generating required shear stress for stem cells in bone tissue engineering. The research findings in this study are expected to provide theoretical guidance to optimize the 3D culture method of seed cells for bone tissue engineering.