GDP-L-fucose is recognized as an important nucleotide sugar and a critical glycosyl donor in fucosylation modification. To establish a new method to synthesize GDP-L-fucose, in this study, a biosynthetic pathway from starch to GDPL-fucose was designed. The pathway is catalyzed by eight enzymes. After these eight enzymes were screened and identified, they were expressed in Escherichia coli and subsequently purified. Based on this, a multi-enzyme cascade system was constructed in vitro to synthesize GDP-L-fucose and the reaction conditions were optimized to enhance its synthesis. The eight enzymes, all derived from thermophilic bacteria, include alpha-glucan phosphorylase (αGP), phosphoglucosamine mutase (PGM), glucose-6-phosphate isomerase (PGI), class I mannose-6-phosphate isomerase (ManA), phosphomannomutase (ManB), mannose-1-phosphate guanylyltransferase (ManC), GDPmannose 4,6-dehydratase (Gmd), and GDP-L-fucose synthetase (WcaG). After optimizing the reaction temperature, pH, enzyme ratio, Mg2+, NADPH concentrations, and phosphate buffer concentration, the reaction was conducted with 1 g/L starch in one vessel, resulting in a GDPL-fucose yield of 53.23% and 0.53 g/L. This study is the first to report the biosynthesis of GDP-L-fucose from starch in vitro, providing a new method for the synthesis of GDP-L-fucose from starch-based renewable resources in vitro.