This is a new expanded method of determining the characterisation of fucoidan from Laminaria japonica (kelp) in rat plasma by high-performance liquid chromatography (HPLC) with fluorescence detection. We tagged fucoidan by fluoresce in isothiocyanate (FITC) for tracking and treated the plasma samples via protein precipitation with 10% trichloroacetic acid and methanol. Column chromatography separation was on a TSK-G4000sw column (7.8 mm × 300 mm, 5 mm) by elution with 0.15 M NaCl. The quantification of fucoidan was performed by HPLC with fluorescence detection. The results suggested that the calibration curve for fucoidan concentration was linear dependent in the limits of 0.5-100μg/mL. The lower limit of quantitation (LLOQ) was 0.5μg/mL and the lower limit of detection (LLOD) was 0.15μg/mL. The intra-day and inter-day precision values were less than 13%and the accuracy ranged from 96.83 to 100.03% at 3 different concentrations. The fucoidan stability of rat plasma at different temperatures and time-points was estimated. The extraction efficiencies ranged from 93.33 to 96.53%and the matrix effect ranged from 92.67 to 95.83%. Method selectivity was evaluated as well. We successfully studied the pharmacokinetic of fucoidan in rat plasma after oral by the validated method. Fucoidan was administered to rats intravenously at a dose of 6 mg/kg and orally at a dose of 20 mg/kg. The Cmax was 7.33μg/mL within 2 h by oral administration; The initial Cmax was 75.59μg/mL. The bioavailability of fucoidan after oral administration to rats was 8.91%.