Fusarielin A (FSA), isolated from culture of Fusarium sp. K432, has anti-fungal/anti-angiogenic activity. Based on our previous structure–activity relationship studies, we prepared FSA-immobilized affinity gel. The results of affinity chromatography with this gel indicated that tubulin and actin are FSA-binding proteins. The binding affinities of tubulin and actin for FSA were estimated by the use of surface plasmon resonance spectroscopy.