This study was conducted on different dairy buffalo herds. A total of 316 buffaloes including 290 buffaloes suffering from reproductive disorders and 26 apparently healthy buffaloes in different rural areas belonging to the Al-Qalyubia Governorate were employed in this study. Epidemiological data revealed that these animals had no history of vaccination against Brucellosis.  Blood sera of these animals were subjected to serological assessments using the Rose Bengal plate test (RBPT), Competitive Enzyme Linked Immunosorbent Assay (cELISA), complement fixation test (CFT) in addition to the use of real-time PCR (rtPCR). MRT and bacteriological examination were performed on milk samples. The obtained results showed that the overall sero-prevalences of brucellosis were recorded as 17.41% , 17.09 % , 16.77% , and 17.09% by RBT, c-ELISA, CFT and rtPCR respectively. B. melitensis biovar 3 was isolated from milk samples 47 (14.87%) It was concluded that for the diagnosis of brucellosis in dairy buffaloes, MRT and RBT remain recommended for screening, CFT is advised for confirming infection in individual animals, and cELISA can be used instead of CFT in some cases. Using rtPCR on serum samples should be regarded as an additional diagnostic technique for detecting and identifying Brucella infection and should be considered in conjunction with other serological tests that can progress and conquer the limits of different immunoassays. According to bacteriological testing, B. melitensis biovar 3 is still the prevalent Brucella field strain found in the milk of buffaloes suffering from reproductive problems.