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Existing studies have not thoroughly investigated the efficacy and safety of sulfonate polymers in animal models and fully elucidate the precise mechanisms by which these polymers act. The activity and safety of sulfonated di\u2010 and triblock copolymers containing poly(sodium styrenesulfonate) (PSSS), poly(sodium 2\u2010acrylamido\u20102\u2010methylpropanesulfonate) (PAMPS), poly(ethylene glycol) (PEG), poly(sodium methacrylate) (PMAAS), poly(acrylic acid) (PAA), and poly(sodium 11\u2010acrylamidoundecanoate) (PAaU) blocks are synthesized and assessed. PSSS\u2010based copolymers exhibit greater anticoagulant activity than PAMPS\u2010based ones. Their activity is mainly affected by the total concentration of sulfonate groups and molecular weight. PEG\u2010containing copolymers demonstrate a better safety profile than PAA\u2010containing ones. The selected copolymer PEG<jats:sub>47<\/jats:sub>\u2010PSSS<jats:sub>32<\/jats:sub> exhibits potent anticoagulant activity in rodents after subcutaneous and intravenous administration. Heparin Binding Copolymer (HBC) completely reverses the anticoagulant activity of polymer in rat and human plasma. No interaction with platelets is observed. Selected copolymer targets mainly factor XII and fibrinogen, and to a lesser extent factors X, IX, VIII, and II, suggesting potential application in blood\u2010contacting biomaterials for anticoagulation purposes. 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