Fucosylated chondroitin sulfate (FCS) oligosaccharides extracted from sea cucumber and depolymerized exhibit potent anticoagulant activity. Knowledge of the antithrombotic activity of different size oligosaccharides and their fucose (Fuc) branch sulfation pattern should promote their development for clinical applications. We prepared highly purified FCS trisaccharide repeating units from hexasaccharide (6-mer) to octadecasaccharide (18-mer), including those with 2,4-disulfated and 3,4-disulfated Fuc branches. All 10 oligosaccharides were identified by their nuclear magnetic resonance structures and ESI-FTMS spectroscopy. In vitro anticoagulant activities and surface plasmon resonance binding tests indicated those of larger molecular sizes and 2,4-disulfated Fuc branches showed stronger anticoagulant effects with respect to anti-FXase activity, as well as stronger binding to FIXa among various clotting proteins. However, both types of FCS 9-mer to 18-mer exhibited molecular size-independent potent antithrombotic activity in vivo at the same dose. In addition, both types of the FCS 6-mer exhibited favorable antithrombotic activity in vivo, although they showed weak anticoagulant activity in vitro. Combining absorption and metabolism studies, we conclude that FCS 9-18 oligomers could remain in the circulation to interact with various clotting proteins to prevent thrombus formation, and appreciable quantities of these oligomers could be excreted through the kidneys. All FCS 9-18 oligomers also resulted in no bleeding, hypotension, or platelet aggregation risk during blood circulation. Thus, FCS 9-18 oligomers with 2,4-disulfated or 3,4-disulfated Fuc branches exhibit potent and safe antithrombotic activity needed for clinical applications.