Abstract: With the ongoing progression of semiconductor integrated circuit manufacturing processes, the reduction in pattern dimensions and the growing complexity of device structures have imposed higher demands on overlay (OV) control within lithography processes. Optical OV measurement results are vulnerable to influences from factors such as underlying film thickness, film characteristics, and OV grating symmetry. As a result, advanced manufacturing processes have increasingly adopted electron beam (E-beam) measurement as a complementary technique. In optical OV measurement, TIS serves as a crucial indicator that reflects the influence of the optical system on OV outcomes, necessitating correction to ensure measurement precision. Nevertheless, research on the impact of TIS in E-beam OV measurement remains scarce. This paper focuses on E-beam OV measurement and utilizes imaging mode variations to verify the presence of TIS effects in E-beam OV measurements. Additionally, the study explores the relationship between TIS effects and E-beam scanning techniques. Moreover, TIS is validated across different layers, and operational approaches are proposed to verify and mitigate TIS during E-beam OV measurement, with the objective of enhancing measurement accuracy.