To identify the existence and composition of the blood-spleen barrier (BSB) in ducks, the microanatomical structures of the duck spleen were investigated by light and transmission electron microscopy, silver staining, enzymatic histochemistry and intravenous injection of ink. The endothelial cells of the sheathed capillaries were cuboidal-shaped and surrounded by an ellipsoid consisting of reticular cells, similar to high endothelial venules (HEVs). After ink injection, carbon particles were initially restricted to the ellipsoid and later trapped in the periellipsoidal lymphatic sheaths (PELS), and then transferred to the periarteriolar lymphatic sheaths (PALS) and splenic nodules over time. Reticular fibers were primarily distributed at the basement membrane of the sheathed capillaries and the periphery of the PELS. Macrophages were primarily distributed at the border between red pulp and PELS. These results suggested that the BSB was present in the ellipsoid and PELS and consisted of the mechanical barrier composed of endothelial cells of the sheathed capillaries, reticular cells and reticular fibers and the biological barrier composed of ellipsoid-associated macrophages. In conclusion, the BSB was identified in the duck spleen for the first time, including cuboidal endothelial cells, ellipsoid-associated macrophages, reticular cells and fibers, and resisting circulating pathogen invasions. The study of BSB in ducks provides a theoretical foundation for the structural composition of the avian immune system.