Background: Diagnosis of Brucellosis is still facing some obstacles; to date it depends on conventional diagnosis based on blood culture and serological methods. PCR method now days offers the hope of rapid diagnosis. Aim:To determine the prevalence of brucellosis among malaria negative febrile patients in Northern kordofan State, Sudan. Methods:In this cross sectional study, one hundred blood samples were collected from febrile malaria negative patients of different ages form North kordofan State during the period from April to May 2016, and examined by Rose Bengal Plate Test (RBPT), ELISA and nested PCR methods. Results:The results showed 40 samples were positive by RBPT method 52 samples were positive by ELISA and 81 sample positive by nested PCR. Conclusion:There was high prevalence of brucellosis among the study population as detected by the three tests. Further studies using various diagnostic methods should be considered to determine the prevalence of human brucellosis at the national level.