Antibodies against β‐glucan, lentinan from “Shiitake” (Lentinus edodes), were raised in the rabbit by subcutaneous immunization. Our antibodies reacted significantly with lentinan by inhibition assay of ELISA. The antibodies did not recognize the other polysaccharides such as amylose, dextran, laminarin and galactan. It was proved that lentinan contents in mushroom could be measured by ELISA with the anti‐lentinan antisera. Its contents were 3.5 mg/g fresh weight in Lentinus edodes. However, lentinan was not contained in Agaricus brazei, Agaricus bisporus and Ramaria bitrytis.