Endo-β-N-acetylglucosaminidase (ENGase) belonging to the glycoside hydrolase family 85 (GH85) is a series of dual-functional enzymes that not only hydrolyze the glycosidic bonds of the N,N′-diacetylchitobiose moieties of N-glycan cores but also transfer chemically synthesized-homogeneous glycans onto glycoproteins. This unique transglycosylation activity enables them to modify proteins with a defined N-glycan structure. In this study, we identified ENGase homolog in Trypanosoma brucei genome by database search. The gene encoding ENGase was cloned into an expression vector with an N-terminal Nus tag, and expressed in Escherichia coli cells. The recombinant ENGase (Nus-Endo Tb) exhibited hydrolytic activity for high-mannose, bi-antennary N-linked oligosaccharides. Such hydrolytic activity can be applied to hydrolyze N-glycans attached to RNase B and human transferrin. Moreover, Nus-Endo Tb also can transfer a sialobiantennary type complex oligosaccharide onto MU-GlcNA, which indicated its transglycosylation activity.