Several biological processes involve glycans, yet understanding their ligand specificities is impeded by their inherent diversity and difficult acquisition. Generating broad synthetic sugar libraries for bioevaluations is a powerful tool in unraveling glycan structural information. In the case of the widely distributed heparan sulfate (HS), however, the 48 theoretical possibilities for its repeating disaccharide call for synthetic approaches that should minimize the effort in an undoubtedly huge undertaking. Here we employed a divergent strategy to afford all 48 HS-based disaccharides from just two orthogonally protected disaccharide precursors. Different combinations and sequence of transformation steps were applied with many downstream intermediates leading up to multiple target products. With the full disaccharide library in hand, affinity screening with fibroblast growth factor-1 (FGF-1) revealed that four of the synthetic sugars bind to FGF-1. The molecular details of the interaction were further clarified through X-ray analysis of the sugar-protein cocrystals. The capability of comprehensive sugar libraries in providing key insights in glycan-ligand interaction is, thus, highlighted.