Carbohydrates on cell surfaces play a crucial role in a wide variety of biological processes, including cell adhesion, recognition and signaling, viral and bacterial infection, inflammation and metastasis. However, owing to the large diversity and complexity of carbohydrate structure and nongenetically synthesis, glycoscience is the least understood field compared with genomics and proteomics. Although the structures and functions of carbohydrates have been investigated by various conventional analysis methods, the distribution and role of carbohydrates in cell membranes remain elusive. This review focuses on the developments and challenges of super-resolution imaging in glycoscience through introduction of imaging principle and the available fluorescent probes for super-resolution imaging, the labeling strategies of carbohydrates, and the recent applications of super-resolution imaging in glycoscience, which will promote the super-resolution imaging technology as a promising tool to provide new insights into the study of glycoscience.