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The aim of this study was to develop a multiplex bead assay using a <i>Brucella</i> rLPS antigen, a <i>Brucella suis</i> smooth antigen, and a <i>Yersinia enterocolitica</i> O:9 antigen that not only discriminates <i>Brucella</i>-infected from <i>Brucella</i>-uninfected pigs and wild boar, but also overcomes the cross reactivity with <i>Y. enterocolitica</i> O:9. Sera from 126 domestic pigs were tested: 29 pigs were <i>Brucella</i> infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 <i>Brucella</i>-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-<i>Brucella</i> infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth <i>B. suis</i> 1330 antigen, all <i>Brucella</i>-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth <i>B. suis</i> antigen and <i>Y. enterocolitica</i> O:9 antigen enabled discriminating all <i>Brucella</i> infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with <i>Y. enterocolitica</i>.