Fucosyl-N-acetylglucosamine disaccharides are present in many biologically important oligosaccharides, such as human milk oligosaccharides, Lewis carbohydrate antigens, and glycans on cell-surface glycoconjugate receptors, and thus have vast potential for infant formulas, prebiotics, and pharmaceutical applications. In this work, in order to screen biocatalysts for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides, we performed sequence analysis of 12 putative and one known alpha-L-fucosidases ofBacteroides fragilisNCTC9343 and constructed a phylogenetic tree of the nine GH29 alpha-L-fucosidases. After that, five GH29A alpha-L-fucosidases were cloned, and four of them were successfully heterogeneous expressed and screened for transglycosylation activity, and a GH29A alpha-L-fucosidase (BF3242) that synthesized a mix of Fuc-alpha-1,3/1,6-GlcNAc disaccharides usingpNP alpha Fuc as donor and GlcNAc as acceptor was characterized. The effects of initial substrate concentration, pH, temperature, and reaction time on its transglycosylation activity were studied in detail. Under the optimum conditions of 0.05 U/mL enzyme, 20 mMpNP alpha Fuc, and 500 mM GlcNAc in sodium buffer (pH 7.5) at 37 degrees C for 45 min, BF3242 efficiently synthesized Fuc-alpha-1,3/1,6-GlcNAc at a maximum yield of 79.0% with the ratio of 0.48 for 1,3/1,6. The molecular dynamics simulation analysis revealed that Loop-4 (His220-Ser245) in the putative 3D model of BF3242 displayed significant changes throughout the thermal simulations, might being responsible for the changes in the ratio of two regioisomeric products at different temperatures. This work provided not only a potential synthetic tool for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides but also a possibility for the formation of regioisomeric products in glycosidase-catalyzed transglycosylation.