Objective To better evaluate the ergonomics of an ankle-foot orthosis (AFO), this study proposed a method for evaluating the impact of AFO stiffness on gait biomechanics based on human-machine coupling model. Methods Firstly, mechanical properties for two kinds of AFO were evaluated, and AFO stiffnesses were quantified. Next, kinetics and kinematics parameters from lower limb joints of 30 subjects were collected during normal walking and walking with two kinds of AFO stiffness. Finally, the impact of AFO stiffness on joint angle,joint torque, and muscle force of lower limbs during walking were quantitatively analyzed by simulation. Results Under the circumstance of two different AFO stiffness, the peak ankle dorsiflexion angle, peak knee flexion angle, and hip extension angle significantly increased (P<0. 05). The peak muscle force of soleus and gastrocnemius also showed an increasing trend. The peak ankle plantarflexion angle, hip flexion angle and peak moment of hip flexion obviously decreased (P<0. 05). Conclusions The AFO human-machine coupling simulation method can effectively realize the quantitative evaluation of the AFO stiffness effect on gait biomechanics. It can also be applied to assess and optimize the ergonomics of other assistive devices. These results provide theoretical guidance for the selection, adaptation, and optimization design of AFO.