The novel calibration method of sub-nanometer accuracy for the line width measurement using STEM images is proposed to calibrate CD-SEM line width measurements. In accordance with the proposed method, the traceability and reference metrology of line width standards are established using Si lattice structures. First, we define the edge of a line as the end of Si lattice structure as the interface between Si lattice and oxide film. Second, an image magnification and inclination angles are calculated using 2D Fourier analysis of a STEM image. Third, the edge positions of the line are detected after the novel noise reduction method using averaging by Si lattice patterns. Then, the uncertainty of the line width measurement is evaluated with the uncertainty contributors of pixel size, edge detections and repeatability. Using the proposed method, the expanded uncertainty less than 0.5 nm for the line width of 45 nm is established.