As an important glycosaminoglycan, keratan sulfate (KS) mainly exists in corneal and cartilage, possessing various biological activities. In this study, we purified KS from blue shark (Prionace glauca) cartilage and prepared KS oligosaccharides (KSO) through keratanase II-catalyzed hydrolysis. The structures of KS and KSO were characterized using multi-dimensional nuclear magnetic resonance (NMR) spectra and liquid chromatography-mass spectrometry (LC-MS). Shark cartilage KS was highly sulfated and modified with ~2.69% N-acetylneuraminic acid (NeuAc) through α(2,3)-linked to galactose. Additionally, KS exhibited binding affinity to Ricinus communis agglutinin I (RCA120) in a concentration-dependent manner, a highly toxic lectin from beans of the castor plant. Furthermore, KSO from dp2 to dp8 bound to RCA120 in the increasing trend while the binding affinity of dp8 was superior to polysaccharide. These results define novel structural features for KS from Prionace glauca cartilage and demonstrate the potential application on ricin-antidote exploitation.