<p>Marine sulfated polysaccharides (SPs) are promising candidates for new anticoagulant drug development, necessitating efficient method to evaluate their anticoagulant activity at the molecular level. In this work, a heparin microarray-based competitive strategy was developed to investigate the specific interactions between SPs and antithrombin Ⅲ (AT). The strategy is based on the principle that SPs competitively bind to the active domain of AT, reducing the signal of AT binding to immobilized heparin on microarrays. Validation was performed using established anticoagulants heparin and enoxaparin as model analytes. After optimizing key experimental conditions, the method successfully determined the IC₅₀ values for three SPs: fucoidan derived from <i>Ascophyllum nodosum</i> (AnF, 50.55 ± 2.79 μg‧mL⁻¹), fucosylated chondroitin sulfate derived from <i>Holothuria tubulosa</i> (FCS_<i>ht</i>, 44.18 ± 4.05 μg‧mL⁻¹), and its selectively degraded sulfation product (S-dFCS_<i>ht</i>, 16.99 ± 6.56 μg‧mL⁻¹). The reliability of these results was confirmed by surface plasmon resonance assay. The strategy’s versatility was further demonstrated by assessing SP interactions with side effect-related proteins, providing valuable insights into both efficacy and safety profiles. Although currently applicable primarily to heparin-interacting anticoagulant targets, this strategy can be extended to other targets by fabricating specialized glycan microarrays, enabling comprehensive evaluation of SP interactions with various anticoagulant targets.</p><p></p>