Aiming at the problem of unclear inter-electrode distance for different depth muscles sEMG signal detection, the effect of inter-electrode distance and muscle fiber depth on sEMG signal in single-fiber sEMG signal simulation model was analyzed firstly in this paper. Then, the relationship between the inter-electrode distance and the amplitude and frequency spectrum of the sEMG signal was analyzed by combining with the sEMG signal acquisition experiment of the extensor carpi ulnaris and diaphragm muscles. The contribution rate of the increase of inter-electrode distance to the increase of sEMG signal amplitude was calculated, and the inter-electrode distance suitable for different muscle levels of sEMG signal detection was determined. Simulation analysis and experimental results show that the increase of inter-electrode can increase the sEMG signal amplitude, and the inter-electrode distance of deeper muscle sEMG signal detection is larger than that of superficial muscle.