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Glycophorins (GPs) in the red blood cell (RBC) membranes of carp (Cyprinus carpio L.) exhibit bacteriostatic activity against various gram-negative and gram-positive bacteria. This antibacterial property is attributed to a sialo-oligosaccharide. For a structural analysis of this O-linked sialo-oligosaccharide, it is necessary to prepare and purify the oligosaccharide from carp GPs. To prepare sialo-oligosaccharides, RBC membrane proteins were solubilized, and anion exchange column chromatography was performed to obtain the GPs. The GP yield from carp RBC membranes was ca.100% as total sialic acid. Then, released oligosaccharides were obtained from GP preparation by β-elimination. Second, the resulting oligosaccharide fraction was separated into two sialo-oligosaccharide components (P-1 and P-2) using the same anion exchange column. The oligosaccharide preparations that contained high amounts of NaCl were desalted with an activated charcoal column combined with ammonium bicarbonate in acetonitrile as an eluent. Using NMR to analyse the structure of the antimicrobial P-1, we determined the alignment of each proton and C1 carbon. To determine the linkage sites, we performed GC-MS using the permethylated P-1. Lastly, we determined that the structure of P-1 was NeuGcα2→6 (Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol.