Disseminated microsporidiosis among HIV/AIDS patients is life-threatening. The incidence may be actually higher than what has been reported. This is due to non-specific presentations of the disseminated cases and also the insensitivity of routine diagnostic technique which contribute to delay in the treatment of the disease. In the present study, we report the use of blood specimens to detect circulating microsporidia DNA, which has not been reported for diagnosis of disseminated microsporidiosis. Blood samples from HIV/AIDSpositive patients were collected over a period of one year. These samples were subjected to PCR assay using species-specific primer EBIEF1/EBIER1. Out of 100 patients, seven were confirmed positive for E. bieneusi by PCR. A fragment of 607 bp was successfully amplified. Identification of circulating microsporidia DNA in blood samples may aid in early diagnosis, thereby allows timely administration of anti-parasitic treatment.