Fucoidan, a fucose-rich polysaccharide from brown algae, has been used for transdermal formulations targeting inflammatory skin conditions, for the treatment of thrombosis, vascular permeability diseases, subcutaneous wounds, and burns. However, the pharmacokinetics of fucoidan after topical application has not been described. In this study, an ointment (OF) containing 15% fucoidan was topically applied to rats at the doses of 50–150 mg/g. The anti-Xa activity was selected as the biomarker, and the amidolytic assay method was validated and applied for pharmacokinetic studies of fucoidan. Fucoidan in OF penetrated the skin and distributed into the skin, striated muscle, and plasma with AUC0–48 = 0.94 μg·h/g, 2.22 μg·h/g, and 1.92 µg·h/mL, respectively. The longest half-life for fucoidan was observed in plasma, then in striated muscle and skin. It was found that the pharmacokinetics of fucoidan after topical OF application was linear, in the range of 50–150 mg/kg. No accumulation of fucoidan in plasma was observed after repeated topical applications of 100 mg/kg during five days. Our results support the rationality of topical application of formulations with fucoidan.