The present study investigates the molecular characteristics of fucoidan obtained from the brown Irish seaweedAscophyllum nodosum, employing hydrothermal-assisted extraction (HAE) followed by a three-step purification protocol. The dried seaweed biomass contained 100.9 mg/g of fucoidan, whereas optimised HAE conditions (solvent, 0.1N HCl; time, 62 min; temperature, 120 °C; and solid to liquid ratio, 1:30 (w/v)) yielded 417.6 mg/g of fucoidan in the crude extract. A three-step purification of the crude extract, involving solvents (ethanol, water, and calcium chloride), molecular weight cut-off filter (MWCO; 10 kDa), and solid-phase extraction (SPE), resulted in 517.1 mg/g, 562.3 mg/g, and 633.2 mg/g of fucoidan (p< 0.05), respectively. In vitro antioxidant activity, as determined by 1,1-diphenyl-2-picryl-hydrazyl radical scavenging and ferric reducing antioxidant power assays, revealed that the crude extract exhibited the highest antioxidant activity compared to the purified fractions, commercial fucoidan, and ascorbic acid standard (p< 0.05). The molecular attributes of biologically active fucoidan-rich MWCO fraction was characterised by quadruple time of flight mass spectrometry and Fourier-transform infrared (FTIR) spectroscopy. The electrospray ionisation mass spectra of purified fucoidan revealed quadruply ([M+4H]4+) and triply ([M+3H]3+) charged fucoidan moieties atm/z1376 andm/z1824, respectively, and confirmed the molecular mass 5444 Da (~5.4 kDa) from multiply charged species. The FTIR analysis of both purified fucoidan and commercial fucoidan standard exhibited O-H, C-H, and S=O stretching which are represented by bands at 3400 cm−1, 2920 cm−1, and 1220–1230 cm−1, respectively. In conclusion, the fucoidan recovered from HAE followed by a three-step purification process was highly purified; however, purification reduced the antioxidant activity compared to the crude extract.