A cross-sectional study was carried out in Matrouh Province. A total of 100 camels and 100 human serum samples were examined serologically for brucellosis using RBPT, BAPAT, and CFT. In addition, multiplex PCR was carried out as further confirmation. The overall prevalence of brucellosis in camels by RBPT, BAPAT, CFT, and PCR were 10, 10, 9 and 9%, respectively while they were 17, 15, 14 and 13% for human samples with non-significant statistical association between them. Molecular characterization of seropositive samples of camels as tested by multiplex PCR clarified that B. abortus was the highest detected Brucella spp. while B. melitensis was the highest detected in humans. Females and older camels were more affected and the highest prevalence rate was observed during winter season. Concerning humans, the results of testing were 17, 15, 14, and 13%, respectively. It was noticed that males were more seropositive than females and age group 20 - 40 years appeared to be the most group at the risk than younger or/and older ones. On studying the effect of locality, it was clear that the highest seroprevalence was recorded in Sallum (26.67%). Finally, winter season showed the highest seasonal prevalence of human brucellosis. Conclusively, brucellosis is alarming in Matrouh Province so there was an urgent need for implementing a proper control program for brucellosis in camels and more attention should be paid towards improving the animal health delivery system in those Provinces that are large in size and share borders with other countries.