The invention provides a stroke calibration method of a mobile robot. The stroke calibration method comprises the steps that the actual stroke of a left wheel and the actual stroke of a right wheel are obtained; first displacement and the turning angle are obtained according to the actual stroke of the left wheel and the actual stroke of the right wheel; the actual diameter of the left wheel and the actual diameter of the right wheel are obtained, and a milemeter is calibrated according to the actual diameter of the left wheel and the actual diameter of the right wheel; second displacement is obtained through an inertia measuring unit; a displacement amount threshold value is obtained; the displacement distance and the displacement amount threshold value are compared; when the displacement distance is smaller than the displacement amount threshold value, an average value of the first displacement and the second displacement is fused; and when the displacement distance is greater than the displacement amount threshold value, fusion is conducted through smoothing. The error correction of the milemeter is achieved, and the system positioning precision is improved.