The establishment of a simple technique to determine the concentration of fucoidan was developed by using a monoclonal antibody against fucoidan. This antibody reacted with fucoidans purified from Laminaria japonica Areschoug (Makombu in Japanese) and Kjellmaniella gyrate Miyabe (Gagome), but not with polysaccharides from Undaria pinnatifida Suringar (Wakame). Neither laminarin nor algenic acid, which are constituents in Laminaria japonica, were recognized by the prepared antibody. Application of the enzymed-linked immunosorbent assay (ELISA) inhibition assay increased the specificity of fucoidan in measuring the fucoidan contents. On the basis of these results, it was ascertained that the ELISA inhibition assay of using the anti-fucoidan monoclonal antibody was rapid, accurate, and sensitive in measuring the content of fucoidan. In addition, the localization of fucoidan in Laminaria japonica was investigated. This is the first report of fucoidan being restricted to the outer cortical layer.