The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.