Whether the lower limb prosthesis can better meet the needs of amputees, the biomimetic performance of the knee joint is particularly important. In this paper, Nokov(metric) optical 3D motion capture system was used to collect motion data of normal human lower limbs, and the motion instantaneous center of multi-gait knee joint was obtained. Taking the error of knee joint motion instantaneous center line as the objective function, a set of six-bar mechanism prosthetic knee joint was designed based on a genetic algorithm. The experimental results show that the movement trajectory of the instantaneous center of the knee joint is basically similar to that of the human knee joint, so it can help amputees complete a variety of gaits and has good biomimetic performance. Gait acquisition technology can provide important data for prosthetic designers and it will be widely used in prosthetic design and other fields.