Background: Brucellosis, a widely spread zoonotic disease, poses significant diagnostic challenges due to its non-specific symptoms and underreporting. Timely and accurate diagnosis is crucial for effective patient management and public health control. However, a comprehensive comparative review of available diagnostic tests is lacking. Methodology/Principal findings: This systematic review addressed the following question: 'What is the accuracy of the available tests to confirm human brucellosis?' Two independent reviewers examined articles published up to January 2023. The review included original studies reporting symptomatic patients with brucellosis suspicion, through any index test, with sensitivity and/or specificity as outcomes. As exclusion criteria were considered: sample size smaller than 10 patients, studies focusing on complicated brucellosis, and those lacking essential information about index or comparator tests. Sensitivity and specificity were assessed, with consideration for the index test, and 'culture' and 'culture and standard tube agglutination test (SAT)' were used as reference standards. Bias assessment and certainty of evidence were carried out using the QUADAS-2 and GRADE tools, respectively. A total of 38 studies reporting diagnostic test performance for human brucellosis were included. However, the evidence available is limited, and significant variability was observed among studies. Regarding the reference test, culture and/or SAT are deemed more appropriate than culture alone. Rose Bengal, IgG/IgM ELISA, and PCR exhibited equally high performances, indicating superior overall diagnostic accuracy, with very low certainty of the evidence. Conclusions/Significance: This systematic review underscores the potential of the Rose Bengal test, IgG/IgM ELISA, and PCR as promising diagnostic tools for brucellosis. However, the successful implementation and recommendations for their use should consider the local context and available resources. The findings highlight the pressing need for standardization, improved reporting, and ongoing advancements in test development to enhance the accuracy and accessibility of brucellosis diagnosis. Author summary: Brucellosis represents a prevalent zoonotic condition that significantly impacts regions constrained by limited resources. Diagnosis, usually based on symptoms and incomplete data, leads to underreporting and delayed treatment. Our comprehensive systematic literature review focused on evaluating the practical effectiveness of current diagnostic approaches for brucellosis, to guide decision-makers. Our analysis involved 38 studies primarily conducted in Asian and African regions, revealing considerable outcome variability. When considering the reference test, culture and/or SAT are deemed more suitable than culture alone. While Rose Bengal, IgG/IgM ELISA, and PCR tests exhibited equally strong performances, the evidence remained notably limited. It is vital to recognize that apart from performance, factors such as accessibility, cost, and ease of use must also be factored into informed decision-making. Our findings emphasize the critical need to expand the scope of validation studies on diagnostic tests and the development of new, more robust, and easily accessible alternatives for addressing brucellosis. This pursuit is essential to meet the urgent demand for enhanced diagnostic capabilities in this field, providing improved methods to combat this challenging disease. [ABSTRACT FROM AUTHOR]