Fatigue failure is one of the most common fracture modes of structural materials in the industrial field. The study of material fatigue mechanisms and methods for predicting fatigue life has always been of significant interest to researchers due to the abrupt and catastrophic failure mode. In recent decades, the performance and functionality of scanning electron microscopy (SEM) have been continuously improved and expanded. Based on this, the development of in situ fatigue testing in SEM has been rapidly developed. This technology plays a crucial role in providing insights into the deformation behavior of materials under fatigue. Keeping this in view, a comprehensive review of the development and application methods of in situ SEM fatigue testing technology is provided here. The development of in situ SEM fatigue testing devices is provided in brief overview, and the application and research progress of this technology in some representative metal structural materials (nickel-based single-crystal superalloys, steel, aluminum alloys and additive manufacturing materials) are analyzed in detail. Moreover, the perspectives on evaluating fatigue damage, particularly about small cracks and the plastic accumulations fatigue behavior, are presented in this study, utilizing the latest advancements in in situ SEM fatigue testing. Remarks about the present and outlook for future work to be done are then provided.