In the present work, bacterial glycosyltransferases are utilized to construct ganglioside glycans in a convergent approach via a sugar.nucleotide regeneration system and one-pot multienzyme reactions. Starting from beta-lactoside enables the diversification of both the glycan moieties and the linkages in the lower alpha-arm and upper beta-arm. Overall, a comprehensive panel of 24 natural a-series (GM3, GM2, GM1a, GD1a, GT1a, and fucosyl-GM1), b-series (GD3, GD2, GD1b, GT1b, and GQ1b), c-series (GT3, GT2, GT1c, GQ1c, and GP1c), alpha-series (GM1 alpha, GD1a alpha, and GT1a alpha), and o-series (GA2, GA1, GM1b, GalNAc-GM1b, and GD1c) ganglioside glycans are prepared, which are suitable for biological studies and further applications. Moreover, a microarray is constructed with these synthesized ganglioside glycans to investigate their binding specificity with recombinant Fc-fused Siglec-7 and Siglec-9, which are immune checkpoint-like glycan recognition proteins on natural killer cells. The microarray binding results reveal that GD3 and GT1a alpha are specific ligands for Siglec-7 and Siglec-9, respectively, and this discovery can lead to the identification of appropriate ligands for investigating the roles of these Siglecs in immunomodulation.