Background: Brucellosis is still considered as one of the major zoonosis afflicting Syrian health and economy. This disease is caused by members of the genus Brucella which are gram-negative bacteria living facultatively within mammalian cells during infection. Objectives: In this paper, a strategy was developed to introduce a new generation of binders called Nanobodies (Nbs) in our combat against Brucella. Nbs are genetically engineered camelid-derived single-domain antibody fragments that are very stable and highly soluble, making them promising tools in numerous biotechnological and medical applications. Materials and Methods: In our previous work, three Nb-displaying phages (Nb-phage), referred to as NbBruc01, 02 and 03, were retrieved by a phage display panning of a Nb library constructed from Brucella- immunized camel. In this work, soluble Nbs were produced after recloning their genes in protein expression plasmid followed by purification with affinity chromatography. Results: Interestingly, two of these soluble Nbs (NbBruc02 and 03) were able to detect Brucella antigens from two main Brucella species (B. abortus and B. melitensis) and distinguish them from those of Yersinia. This is remarkable as the camel IgG failed in such antigen discriminations. High similarity, mainly in the structure of lipopolysaccharides (LPS) of these different types of bacteria, causes regular serum cross reactivity and thus lack of specificity urging the need for more accurate diagnostic techniques, e.g. a multiplex PCR. Furthermore, NbBruc02 and 03 targets may represent Brucella immunodominant proteins as shown by immunoblotting. Conclusions: In addition to their own importance, identifying these antigenic targets will open new perspectives for diagnosis, vaccines and treatment of Brucellosis.