To investigate the structural properties and catalytic activity of α-L-fucosidases belonging to the GH29A family, the gene coding for the AlfB enzyme from Lactobacillus rhamnosus GG was obtained from the NCBI database. A bioinformatics analysis was conducted on AlfB, followed by heterologous expression of the recombinant enzyme in Escherichia coli under optimized conditions. The bioinformatics analysis revealed that AlfB was a single-domain enzyme with two conserved active sites: the nucleophilic catalytic residue Asp166 and the acid-base catalytic residue Glu32. The optimal induction conditions for the recombinant enzyme were determined to be 25 ℃ for a duration of 28 h. The optimal temperature and pH of the purified recombinant enzyme was 35 ℃ and 6.0, respectively. It was strongly inhibited by Cu²⁺ but strongly activated by Mn²⁺. Recombinant AlfB exhibited a high affinity for 2'-fucosyllactose (2'-FL) and transformed p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and lactose into 2'-FL and its isomer 3'-FL via transglycosylation. These results set the stage for further elucidating the catalytic activity and mechanism of action of AlfB.