Abstract:β-N-Acetylhexosaminidases (EC.3.2.1.52) are an important class of glycosidases that catalyze hydrolysis of terminal N-acetyl-β-D-hexosamine from various oligosaccharides and polysaccharides. These enzymes are widely distributed in microorganisms, plants, and animals, and play crucial roles in nature. Some β-N-acetylhexosaminidases can catalyze glycosyl transfer to form β-N-acetyl-D-hexosaminides, which have shown great potentiality in enzymatic synthesis of functional glycans. In this review, catalytic mechanisms and biological functions of β-N-acetylhexosaminidases, and potential applications of these enzymes in the synthesis of β-N-acetyl-D-hexosaminide are summarized.