Objective To analyze muscle pre-activation and surface electromyography (sEMG) characteristics of knee and ankle joints of long-term Tai Chi practitioners during brush-knee twist-step and normal walking, and explore the neuromuscular control strategies of Tai Chi to prevent falls. Methods Vicon motion capture system, Kistler force plate, and Noraxon sEMG system were synchronously used to collect the EMG signals of the rectus femoris, biceps femoris, tibialis anterior muscle, lateral head of gastrocnemius and body posture information during brush-knee twist-step and normal walking. The pre-activation and co-contraction of knee and ankle joints were calculated by integrated EMG of the rectus femoris/biceps femoris, tibial anterior/lateral gastrocnemius muscles. Results Compared with normal walking, the average time of brush-knee twist-step in four phases was significantly increased. There was a significant difference in the percentage of time in four phases. The knee joint co-contraction level and pre-activation level decreased, and the ankle joint co-contraction level and pre-activation level increased. Conclusions Long-term Tai Chi exercises may increase the activation level of the muscles around knee joints and enhance the synergy in muscle groups to help stabilize the joint. The results provide references for rehabilitation assessment and training of neuromuscular control disorders.