The disaccharide galactose-β1,3-N-acetylgalactosamine (Galβ1,3-GalNAc) attached to serine and/or threonine residues of proteins, also known as the Thomsen-Friedenreich (TF) antigen, is highly expressed in various types of human carcinomas. It has been shown to contribute to tumor development, progression, and metastasis. However, current methods have limited power in detecting and imaging TF antigens among a variety of complex cell-surface glycans. Here we describe a tandem enzymatic strategy to detect and label TF antigen disaccharide with high sensitivity and selectivity. We demonstrate that this strategy enables detection of TF antigens on proteins, profiling and identification of unknown TF antigen-modified glycoproteins, and simultaneous labeling of multiple forms of complex glycan motifs on the same cell. This approach expands the capability of glycan labeling to probe the functional role of TF antigens in cancer biology.