The glycocalyx is a dense and diverse coat of glycans and glycoconjugates responsible for maintaining cell surface integrity and regulating the interaction of cells with the external environment. Transmembrane mucins such as MUC1 and MUC16 comprise a major component of the epithelial glycocalyx and are currently used to monitor disease progression in cancer. At the ocular surface, multiple lines of evidence indicate that abnormal expression of the enzymes responsible for glycan biosynthesis during pathological conditions impairs the glycosylation of transmembrane mucins. It is now becoming clear that these changes contribute to modify the interaction of mucins with galectin-3, a multimeric lectin crucial for preserving the ocular surface epithelial barrier. This review highlights the potential of using the epithelial glycocalyx as a reliable source for the generation of biomarkers to diagnose and monitor ocular surface disease.