Brucellosis is one of the most important diseases affecting both humans and animals in most developing countries. This study aimed to evaluate the efficiency of different serological tests used for the diagnosis of Brucellosis on serum samples as well as immunoassays adapted to detect anti-Brucella antibodies in the milk whey. Besides, seeking the efficiency of the conventional bacteriological examination against Real-time PCR for the direct detection of Brucella species in milk samples. A total of 105 cows that belonged to a private farm in the Giza governorate were selected.  Out of these 105 cows, 65 cows were suffering from reproductive disorders, and the remaining 40 cows were in contact with animals. This farm had no history of brucellosis vaccination and was under brucellosis quarantine measurements. Out of the examined 105 serum samples by the serological tests, 28 (26.7%), 25 (23.8%), 20 (19%), and 23 (22%) cows' sera were reacted positively to the Buffered Acidified Plate Agglutination Test (BAPAT), Rose Bengal plate test (RBPT), Rivanol test (RIV.T) and Complement Fixation Test (CFT) respectively. On the corresponding picture, out of 105 whey (w) samples examined by wBAPAT, wRBPT, and wRiv.T, antibodies against Brucella were detected in 17 samples (16.2%), 14 samples (13.3%), and 11 samples (10.5%) by these tests respectively. The percent positive result of the examined milk samples was 20% by MRT (21 out of 105 milk samples), while milk-ELISA (m-ELISA) showed 18.1% positive results to Brucellosis (19 out of 105 milk samples). The real-time PCR identified Brucella melitensis in 12 milk samples out of the examined 105 cows' milk samples (11.4%). On the contrary, the conventional culture method identified nine (8.5%) Brucella. In conclusion, real-time PCR is sensitive, safe, and efficient for the direct detection of Brucella species in Milk samples. The serological tests identified a higher number of reactors than the corresponding whey serological tests.