The detailed chemical structure and chain conformation of a novel fucoidan from the sea cucumber Stichopus chloronotus (Fuc-Sc) were investigated by HPLC, IR, NMR spectroscopy and multi-angle light scattering. It mainly consisted of l-fucose and sulfate esters at mole ratio of 1.0:1.3 and contained a →3)-α-l-fucose-2S-(1→ linear backbone. Besides, the chain conformation parameters α(η) (0.658?±?0.001) and α(h) (0.612?±?0.002) indicated that Fuc-Sc exhibited a random coil conformation in PBS. The worm-like cylinder model parameters M(L) (526?g/mol?nm) and q (6.9?nm) confirmed that Fuc-Sc adopted a flexible chain. Fuc-Sc possessed negative charges and good thermal stability. Moreover, Fuc-Sc showed significant inhibition of lipid peroxidation and obvious activation effects on RAW 264.7 cells by promoting their proliferation and the production of NO and TNF-α. The present study facilitated further understanding of the structure-activity relationship of Fuc-Sc and its application in food and pharmacy industries.