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Though serodiagnosis of actinomycetoma is established, that of eumycetoma due to Madurella mycetomatis is limited because of lack of pure antigen. Reliable rapid tests are needed to make an accurate timely diagnosis. The purpose of this study is to detect antigen parts of M. mycetomatis, which act specifically with M. mycetomatis antibodies.Cytoplasmic antigen was prepared from molecularly identified cultures of M. mycetomatis by sonication, ultracentrifugation, dried, weighed and appropriately reconstituted. M. mycetomatis cytoplasmic antigen were separated using 12% sodium dodecyl sulfate-polyacrylamide gel, and immunoblotting to detect the reactive ones.Immunoblotting was carried out in nitrocellulose strips containing different molecular size. Sera from patients and co-patients as control were used.When stained with Coomassie brilliant blue R 250 seven molecular weights appeared but only three, 45, 60, 95 kDa reacted with M. mycetomatis patients few from control group, one from a malaria patient. No reactive band was observed with sera from actinomycetoma, Aspergillus flavus-associated aspergillosis, schistosomiasis, leishmaniasis, fungal sinusitis nor healthy controls.Specific fractions of M. mycetomatis antigen which were demonstrated by immunoblotting showed 75% sensitivity and 95% specificity. The true negative tests were 14 patients (32.5%). This also means that immunoblotting is reasonably reliable in diagnosis and follow-up of eumycetoma patients.