Abstract: Fucosidases play a key role in the biodegradation and functional exploration of fucoidan, yet their catalytic diversity and molecular mechanisms remain unclear. This study aims to explore novel fucosidases from the marine microorganism Pontiella sulfatireligans, and to analyze their enzymatic characteristics and structural basis. The discovery of glycosidases was conducted through genomic comparison and annotation to identify enzymes involved in fucoidan degradation. The candidateα-L-fucosidase gene, PSf1, was cloned and expressed, and its hydrolytic activity and substrate affinity were measured using p-nitrophenyl fucoside as a substrate. Substrate specificity was assessed through a substrate spectrum analysis. The three-dimensional structure of PSf1 was resolved using X-ray crystallography, and molecular dynamics simulations were performed to analyze its binding mode and stability differences with different substrates. The results showed that when using pNP-Gal as a substrate, PSf1 exhibited a hydrolytic activity of 3 U/mg, with a K_m value of 337 μmol/Lol/L. Substrate specificity analysis revealed for the first time that PSf1 has dual activity, hydrolyzing both α-fucosidic bonds and β-galactosidic bonds. Structural analysis(2.8 Å, PDB ID: 8Z63)showed that the enzyme exists as a non-mirrored dimer in its active form, where the fucose moiety of the substrate and its structural analog, galactose, bind within the conserved active site via hydrogen bonds and other interactions. Molecular dynamics simulations further demonstrated that PSf1 exhibits higher binding stability and lower binding free energy for the fucose substrate, while its hydrolytic activity towards galactosidic bonds originates from recognition of structural similarity. PSf1 is a multifunctional glycoside hydrolase with both α-fucosidase and β-galactosidase activity. The discovery of this enzyme not only elucidates the metabolic mechanism by which P. sulfatireligans efficiently utilizes fucoidan, but also provides a novel enzymatic resource and theoretical foundation for the functional development of fucoidan and enzyme-based synthesis and modification of human milk oligosaccharides, with significant potential applications.