Glycocalyx refers to the outermost layer or sugar cell coat of many cells, which consists of glycoproteins, glycolipids, and glycoRNAs. It often plays a critical role in many biological processes, such as mediating immune responses and intercellular communications. The drastically altered glycosylation pattern in cancer cells, which results in a different sugar coat than healthy cells, has been observed for decades. It has been demonstrated to be correlated with tumor growth, proliferation, invasion, metastasis, and immunity. A comprehensive understanding of the glycocalyx differences between healthy cells and cancerous cells will be instrumental for the development of faster and more effective cancer diagnoses and therapies. We envisioned to incorporate advanced high-resolution microscopy and glycan-binding protein engineering for better imaging techniques in studying cancer-associated cell surface glycans. This thesis summarizes in detail our recent advancements in developing a molecular toolkit suitable for cell surface glycan imaging with expansion microscopy. In this toolkit, engineered glycan-binding proteins are selected as probes for glycan imaging to achieve structural specificity. Sortase-mediated transpeptidation is used as a versatile, site-specific protein modification method that can be used for different kinds of glycan-binding proteins, including carbohydrate-binding modules (CBMs) and adaptive immune proteins, while maintaining their binding affinities and specificities.