Brucellosis, a reported zoonotic disease caused by several species of the genus Brucella, is endemic to many countries, including Morocco, and poses a major public health challenge. Available studies and national surveillance data highlight its persistent presence and impact, with an incidence rate that is difficult to estimate owing to under-reporting of cases. Human diagnosis is mainly based on serological tests (Rose Bengal and ELISA) without the routine implementation of molecular techniques, which are faster and more specific. To our knowledge, in Morocco, no study has demonstrated the use of polymerase chain reaction (PCR) as a routine diagnostic tool. This study aimed to develop, test, and optimise a multiplex real-time PCR assay capable of detecting all Brucella spp. using the conserved bcsp31 gene to differentiate between B. melitensis and B. abortus. Using the KoMa plasmid as an internal extraction control, this study assessed the analytical sensitivity of the assay, which showed a detection limit of ≤10 plasmid copies and 5-10 genomic copies per reaction, including its specificity, which reached 100%, with no amplification observed for non-Brucella strains. This pilot study evaluated the diagnostic performance of the assay using 30 suspected brucellosis samples. All 17 were confirmed to be positive by ELISA and Rose Bengal tests, whereas 13 were negative. Seventeen positive samples were confirmed by real-time PCR, which showed 100% agreement (95% confidence interval [CI]: 98%). The results showed that all positive human cases studied were caused by B. melitensis. This fast and reliable method is a promising tool for monitoring and managing the rapid clinical diagnosis of human brucellosis. However, validation on a larger scale is required.