Glycans, also known as carbohydrates, are one of the three fundamental building blocks of life. Glycans are often attached to proteins and lipids on cell surfaces and form a dense layer of matrix, called glycocalyx. Glycans are believed to encode the structural organization of the glycocalyx, and in doing so, tune how these systems perceive and process critical information from the microenvironment. Notably, the glycocalyx undergoes changes with the onset of cancer and the altered glycans play functional roles in tumor growth and metastasis. However, a deficit of high-quality tools for molecular recognition of glycans, particularly antibodies, presents a significant barrier to advancing the field of glycoscience. In my dissertation work, I address two key bottlenecks in the development of anti-glycan antibodies. First, existing anti-glycan antibodies often exhibit low affinity, and conventional affinity maturation techniques frequently compromise specificity. To overcome this, I combined structure-guided rational design with directed evolution to systematically analyze the effects of mutations on antibody affinity and specificity. This approach enabled me to engineer affinity-matured antibodies that retain their specificity. Second, the discovery of anti-glycan antibodies is inherently difficult due to the weak immunogenicity of glycans and the challenges associated with synthesizing pure glycan structures. To address this, I utilized glycosylated bacterial outer membrane vesicles (glycoMVs) to modulate the immune response against specific glycan structures. This strategy allowed me to successfully isolate antibodies targeting multiple distinct glycans. Additionally, I explored two complementary antibody isolation techniques: traditional yeast surface display (YSD) and next-generation sequencing (NGS). Both approaches led to the discovery of high affinity, high specific anti-glycan antibodies. Collectively, my work advances the development of high-affinity, highly specific anti-glycan antibodies, paving the way for improved research tools and potential therapeutic applications in glycoscience.