O-GlcNAcylation is an O-linked β-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from β-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.