Fucosyllactose (2′-FL), one of the most commercially valuable human milk oligosaccharides, relies on an efficient α-1,2-fucosyltransferase (FucT) for its biosynthesis. However, existing FucT commonly suffer from low heterologous expression levels, poor solubility, and insufficient catalytic activity, which constitute critical bottlenecks restricting the large-scale synthesis of 2′-FL. In this study, an FucT from Helicobacter pylori (H.p-FucT) was used as a probe to mine novel FucT genes from the genomes of Helicobacter species. Through homologous sequence alignment and phylogenetic analysis, three candidate FucT-encoding genes were identified. These three candidate genes, along with the template gene, were recombinantly expressed, and their soluble expression levels and catalytic activities were comprehensively compared. Among them, the FucT from Helicobacter mustelae (H.m-FucT) was selected for further enhancement of soluble expression via molecular chaperone co-expression. Five molecular chaperone co-expression systems were constructed, among which pKJE7 exhibited the most pronounced solubilizing effect, increasing the soluble expression level of H.m-FucT by approximately 6-fold compared with the control group without co-expression. Subsequently, a bifunctional enzyme FKP and polyphosphate kinase PPK2 were co-expressed to establish an in vitro multi-enzyme cascade catalytic system encompassing fucose activation, transglycosylation, and cofactor recycling. Through single-factor optimization combined with a fed-batch strategy, 2′-FL was produced, reaching a titer of 3.40g/L. This study aimed to expand the sources of FucT and enhance its soluble expression level, with the goal of providing enzyme resources and technical support for the efficient synthesis of 2′-FL and other fucosylated human milk oligosaccharides.