A heparin glycan chip (HepGlyChip) with a 4800-fold enhanced signal-to-noise ratio as compared with the control without heparin was developed for high-throughput analysis of heparin-protein interactions for new drug development and for screening biological samples in diagnostic applications. As a proof of concept, a heparin glycan microarray was prepared on a poly(styrene-co-maleic anhydride) (PS-MA)-coated glass slide. Heparin was covalently immobilized on poly-l-lysine (PLL) layer with multiple binding sites by sulfo-ethylene glycol bis(succinimidylsuccinate) (sulfo-EGS), increasing the signal-to-noise ratio, minimizing nonspecific binding of target proteins, and resulting in a three-dimensional (3D) structure on the HepGlyChip. This on-chip signal amplification platform was successfully demonstrated by probing the heparin microarray with the highly specific heparin-binding protein antithrombin III (AT III).