The diffraction limit prevents traditional optical imaging methods from observing organelle structures and interaction between the organelles. As the imaging technique with the highest resolution among the three super-resolution techniques, single-molecule localization microscopy is an important tool for research in the field of life science. Single-molecule imaging technology with a large field of view and high throughput has been widely used in the biomedical field to observe and analyze complex biological structures and functions due to its high resolution, wide imaging range, and short imaging time. In this paper, the recent research progress of high-throughput single-molecule localization microscopy with a large field of view is reviewed from four perspectives: hardware scanning-based mosaic imaging technology, large-array sCMOS large-field-of-view high-throughput imaging technology, large-depth single-molecule positioning imaging technology, and high-throughput data analysis technology. Finally, the development direction for the large-field-of-view high-throughput single-molecule localization microscopy is prospected.