The intrinsic fluorescence intensity of Fusarium solani lectin was quenched upon binding to mono- and oligosaccharides without any change in the emission maximum and it was used to determine association constants for several sugars and glycans. The lectin interacted very poorly with monosaccharides but well with disaccharides (T-antigen and LacNAc) with a distinction between β1→4 and β1→3 linkages. Among the monosaccharides, the interaction was observed only with Gal/GalNAc derivatives and not with Glc/Man derivatives. Thermodynamic studies revealed that the binding of the lectin with all the saccharides is enthalpically driven and exothermic in nature. Asialo-triantennary N-glycan and asialo-biantennary N-glycan showed higher affinity than monovalent LacNAc with significant increase in binding enthalpy, pointing towards the importance of multivalency in the lectin–ligand interactions. Time-resolved fluorescence measurement indicated the lectin has two lifetimes for tryptophan and the shorter lifetime is affected on ligand binding.