Abstract: As an important analytical tool at the nano-scale, the accuracy of the length measurement value of the transmission electron microscopy (TEM) will directly affect the measurement results of samples. In this study, the length measurement error and length measurement repeatability of the high-resolution TEM were calibrated using single-crystal silicon lattice standards, and the uncertainty of the length measurement error was evaluated. To illustrate the applicability of the calibration method, calibration experiments were conducted on three different models of high-resolution TEMs in the study. The results show that the calibration method is widely representative and can achieve an accurate evaluation of TEMs with measured quantities traceable to the silicon lattice constant, which provides technical assurance for accurate measurements in the field of nanotechnology.