Technical Abstract: One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136 kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus eduliswas compared and the results showed that none of them were positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5-7 µm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong, positive signals on cytoplasm of some spherical cells with diameter of 7-8 µm. Some other cells with higher nucelo-plasmic ratio, about 5-6 µm in diameter showed weak positive signals on membranes. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive.