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α- l -Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α- l -Fucosidases, glycoside hydrolases for catalyzing the removal of α- l -fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α- l -fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α- l -fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α- l -fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α- l -fucosidases. [ABSTRACT FROM AUTHOR]