Bovine brucellosis is a disease of global socio-economic importance caused by Brucella abortus. Diagnosis is mainly based on bacterial culture and serology. However, these methods often lack sensitivity and specificity. A range of molecular diagnostic methods has been developed to address these challenges. Therefore, this study aims to investigate the diagnostic accuracy of molecular tools, in comparison to gold standard bacterial isolation and serological assays for the diagnosis of bovine brucellosis. The systematic review and meta-analysis were conducted based on analyses of peer-reviewed journal articles published between January 1, 1990, and June 6, 2020, in the PubMed, Science Direct, Scopus, and Springer Link databases. Data were extracted from studies reporting the use of molecular diagnostic methods for the detection of B. abortus infections in animals according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. The quality of included journal articles was assessed using the quality assessment of diagnostic-accuracy studies assessment tool and meta-analysis was carried out using Review Manager. From a total of 177 studies, only 26 articles met the inclusion criteria based on PRISMA guidelines. Data from 35 complete studies were included in the meta-analysis and used to construct 2 × 2 contingency tables. Improved diagnostic performance was observed when tissue (sensitivity 92.7% [95% confidence interval (CI) 82.0-98.0%]) and serum samples (sensitivity 91.3% [95% CI 86.0-95.0%]) were used, while the BruAb2_0168 locus was the gene of preference for optimal assay performance (sensitivity 92.3% [95% CI 87.0-96.0%] and specificity 99.3% [95% CI 98.0-100.0%]). Loop-mediated isothermal amplification (LAMP) had a higher diagnostic accuracy than polymerase chain reaction (PCR) and real-time quantitative PCR with sensitivity of 92.0% (95% CI 78.0-98.0%) and specificity of 100.0% (95% CI 97.0-100.0%). The findings of this study assign superior diagnostic performance in the detection of B. abortus to LAMP. However, due to limitations associated with decreased specificity and a limited number of published articles on LAMP, the alternative use of PCR-based assays including those reported in literature is recommended while the use of LAMP for the detection of bovine brucellosis gains traction and should be evaluated more comprehensively in future.