Terminal N-acetylglucosamine (GlcNAc) N-linked glycosylation is a truncated N-glycosylated modification that has been reported to be involved in various diseases, such as autoimmune diseases, cancers, and neurodegenerative diseases. New and simple tools will be always valuable for further characterization of the functions of this kind of glycosylation. Our previous paper proved that an optimized lectin created from Agrocybe aegerita GlcNAc selective lectin (AANL) named AANL6, can effectively identify O-GlcNAcylation, which is terminal GlcNAc O-linked glycosylation. We speculated that AANL6 could also be used to identify terminal GlcNAc N-linked glycosylation. Using therapeutic monoclonal antibodies as a model of terminal GlcNAc N-glycosylated proteins, we proved that AANL6 could selectively identify terminal GlcNAc N-linked glycosylation. The ratio of terminal GlcNAc N-linked glycosylation was increased by enrichment with AANL6 in human serum. Using cell membrane proteins as a complex sample, we found that AANL6 bound to the sperm surface, which expresses abundant terminal GlcNAc N-glycans, but did not bind to some tumor cell surfaces such A549 and MCF-7 cells, which is rich in high mannose glycoforms. In conclusion, AANL6 was identified as a powerful tool to probe terminal GlcNAc N-linked glycosylation and would be valuable for uncovering the function of this glycosylation.Copyright © 2023. Published by Elsevier Ltd.