Muscle fatigue has widespread application in the field of rehabilitation medicine. The paper studies the muscle fatigue using surface electromyogram (sEMG) in the background of rehabilitation training system. The sEMG and ventilatory threshold of vastus lateralis, rectus femoris and erector spinae are collected synchronously and the electromyogram fatigue threshold (EMGFT) of different sEMG was analyzed by increasing load cycling experiments of 10 healthy subjects. This paper also analyzes the effect of isotonic and isometric contraction on EMGFT. Results showed that the appeared time of EMGFT was earlier than that of ventilatory threshold in the incremental load cycling. While the differences were subtle and EMGFT was verified to be effective. EMGFT has been proven effective for different muscle contraction by comparing the EMGFT of vastus lateralis and erector spinae. EMGFT could be used to keep muscle injuries from overtraining in the process of rehabilitation. Therefore, EMGFT has a great significance for femoral shaft fractures’s fatigue monitoring in rehabilitation training.