Abnormal glycosylation is a hallmark of tumor development, and tumor-associated carbohydrate antigens are potential immune targets for tumor therapy. Tumor-associated extracellular microvesicles are subcellular vesicles released from cell membranes that have immunogenicity similar to that of precursor cells. However, unmodified tumor-derived microvesicles have weaknesses, such as low immunogenicity, poor biostability, and short half-life in vivo. For the first time, we herein generated extracellular microvesicles containing modified tumor-associated carbohydrate antigens by constructing a cell line with highly expressed antigen-processing enzymes utilizing fluorine-modified monosaccharide substrates via a metabolic oligosaccharide engineering strategy. The microvesicles were applied to tumor immunity, achieving enhanced immunoprophylaxis and immunotherapy effects. Furthermore, the mechanisms of antitumor immunity were explored. Our findings may provide new insights into the adhibition of suitably modified extracellular microvesicles and the development of more effective carbohydrate-based anticancer vaccines.