Vibrio splendidus and -the two pathogens of the skin ulcer syndrome of sea cucumber were prepared with rabbits.Cross-reactions of antisera with other 11 main marine pathogens were detected by agglutinationand the results showed low cross-reaction which was eliminated by heightening the dilution of antisera. The cross reaction assay and absorb assay indicated the method was successful in distinguishing those pathogens. Working concentration of two antisera and FITC-goat-anti-rabbit IgG were assayed by grads dilutionsKH2 and FITC-IgG were 400KH2 and FITC-IgG were 200100 and 80 respectively for detecting frozen section of tissues. The sensitivity of the method was 2.4×10^{cells/mL. An artificial infection experiment on sea cucumber by 2 pathogens were carried out，and frzoen sections of tissues were detected by IFAT. Results showed the established method could detect the pathogens from diseased sea cucumberV. splendidus and were stained with bright yellow-green. Positive cells in frozen sections indicated that the mouth tumidity and skin ulceration symptoms of diseased sea cucumber were caused by the invading of bacteria. The overall detecting results suggest that applying this technique properly in clinical diagnosis of skin ulcer syndrome of}