Galectins exhibit multiple roles through recognition of diverse structures of β-galactosides. However, this broad specificity often hinders their practical use as probes. In the present study we report a dramatic improvement in the carbohydrate specificity of a multi-specific fungal galectin from the mushroom Agrocybe cylindricea, which binds not only to simple β-galactosides, but also to their derivatives. Site-directed mutagenesis targeting five residues involved in β-galactose binding revealed that replacement of Asn46 with alanine (N46A) increased the binding to GalNAcα1-3Galβ-containing glycans, while eliminating binding to all other β-galactosides, as shown by glycoconjugate microarray analysis. Quantitative analysis by frontal affinity chromatography showed that the mutant N46A had enhanced affinity towards blood group A tetraose (type 2), A hexaose (type 1) and Forssman pentasaccharide with dissociation constants of 5.0 × 10⁻⁶ M, 3.8 × 10⁻⁶ M and 1.0 × 10⁻⁵ M respectively. Surprisingly, all the other mutants generated by saturation mutagenesis of Asn46 exhibited essentially the same specificity as N46A. Moreover, alanine substitution for Pro45, which forms the cis-conformation upon β-galactose binding, exhibited the same specificity as N46A. From a practical viewpoint, the derived N46A mutant proved to be unique as a specific probe to detect GalNAcα1-3Galβ-containing glycans by methods such as flow cytometry, cell staining and lectin microarray.