Endo-β-N-acetylglucosaminidases (ENGases) are essential enzymes for hydrolyzing N-glycans, with applications in protein N-glycosylation analysis and glycoprotein synthesis. In this study, a novel GH18 family ENGase, Endo-LB, was identified from Listeria booriae FSL A5-0281. Composed of 593 amino acids (65.78 kDa), Endo-LB features with two domains: an Endo S-like catalytic domain and a mucin-binding protein (MucBP) domain. Recombinant Endo-LB, expressed in Escherichia coli BL21 (DE3) pLysS, exhibited a specific activity of 198.25 U/mg and hydrolyzed high mannose-type N-glycans at a temperature from 4 °C to 60 °C with optimal activity at 37 °C and pH 6.0 (range 3.0 to 10.0), making it versatile for various environmental conditions. The MucBP domain does not affect soluble Endo-LB activity but influences interaction with mucin on cell surface, suggesting potential application in targeting specific glycoproteins in complex biological environments. To address the heterogeneity of N-glycans in Pichia pastoris (Komagataella phaffii) expression, Endo-LB was further expressed in the Golgi of P. pastoris, efficiently producing glycoproteins, such as Erythropoietin (EPO) (37 mg/L) and Darbepoetin α (53 mg/L) with nearly complete N-glycans truncation, which can be further extended to generate diverse N-glycan structures. These findings highlight the versatility and potential utility of Endo-LB in glycoprotein engineering and biotechnological applications.