Heparan sulfate (HS) is a non-immunogenic antigen, and developing antibodies against specific sulfated patterns in HS poses significant challenges. Herein, we employed an innovative immunization strategy that exploits the molecular mimicry of HS to generate antibodies against HS sequences. Mice were immunized with synthetic sulfated oligo-l-idose (ID49) that mimics optimum 67% of the conserved structure of HS ligands. This immunization of ID49@CRM197 with alum and Freund's adjuvant resulted in the production of robust IgG antibody responses targeting ID49 and cross-reactivity with the N-sulfated HS ligands compared to N-unsubstituted and N-acetate domain synthetic HS ligands. Such a pharmacological agent exhibited distinct staining of tissue sections and cell lines and induced complement-dependent cell cytotoxicity against SK-BR-3 cancer cells. Moreover, these antibodies inhibited heparin-mediated anticoagulation activity similar to that of protamine. These findings highlight the biomarker and possible therapeutic capability of the antibodies.