Sialosides containing (oligo-)N-acetyllactosamine (LacNAc, Galβ(1,4)GlcNAc) as core structure are known to serve as ligands for Siglecs. However, the role of tandem inner epitope for Siglec interaction has never been reported. Herein, we report the effect of internal glycan (by length and type) on the binding affinity and describe a simple and efficient chemo-enzymatic sugar nucleotide regeneration protocol for the preparative-scale synthesis of oligo-LacNAcs by the sequential use of β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosyl transferase (β3GlcNAcT). Further modification of these oligo-LacNAcs was performed in one-pot enzymatic synthesis to yield sialylated and/or fucosylated analogs. A glycan library of 23 different sialosides containing various LacNAc lengths or Lac core with natural/unnatural sialylation and/or fucosylation was synthesized. These glycans were used to fabricate a glycan microarray that was utilized to screen glycan binding preferences against five different Siglecs (2, 7, 9, 14 and 15).