Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups. The labeled O-GlcNAc glycopeptides could be efficiently enriched based on the equilibrium between the hydrazine and oxime bonds. Good specificity of the glycopeptide enrichment was observed from the mixtures of glycopeptide and non-glycopeptides using the CHO-GlcNAc method. Then, it was applied to analyze O-GlcNAcylation in the nucleus of HeLa cells, and 829 potential O-GlcNAcylation sites on 274 glycoproteins were identified, including the two readers of m6A (YTHDF1 and YTHDF3), which could provide clues for the mechanism of crosstalk between O-GlcNAcylation and other PTMs of proteins and RNA. Thus, this method could be a versatile tool for the proteomic analysis of O-GlcNAcylation.