α2,6-Sialylated glycans are crucial molecular targets for cancer diagnosis and clinical research. In this work, a novel ultrasensitive electrochemical biosensor was fabricated based on a graphite oxide (GO), Prussian blue (PB), and PTC-NH 2 (an ammonolysis product of 3,4,9,10-perylenetetracarboxylic dianhydride) nanocomposite for the selective detection of α2,6-sialylated glycans. To increase the sensitivity of the electrochemical biosensor, gold nanoparticles (GNPs) were immobilized on a GO–PB–PTC-NH 2 modified glassy carbon electrode (GCE). Sambucus nigra agglutinins (SNAs), which specifically bind with α2,6-sialylated glycans, were covalently immobilized on GNPs for the sensitive detection of α2,6-sialylated glycans in serum. This proposed method can be applied to human serum, and it worked well over a broad linear range (0.1pgmL −1 –500ngmL −1 ) with detection limits of 0.03pgmL −1 . Moreover, recovery of the spiked samples ranged from 100.2% to 105.0%, suggesting that this excellent electrochemical biosensor can be used for the practical detection of α2,6-sialylated glycans.