Background: Brucellosis is continuing to be a major public health concern globally; diagnosis of brucellosis should be consistent with the laboratory test and clinical appearance. Therefore; anew serological test must be easy in applied and the results can be obtained within a short time. Aim of study: This study amid to establish a highly accurate universal diagnostic test detect brucellosis by microtiter in less than one hour, read the result by naked eye. Material and methods: The study was conducted in Fakous and Zagazig Fever Hospital and some hospitals in (Egypt), 2020 to 2023. Out of (1292) patients diagnosed for clinical brucellosis. 323 healthy case of different age and sex were also studied as control. All participants were clinical examination, and lab investigations. Result: Among (1292) clinically suspected cases (54%) females, (46%) males. Clinically have a fever case with brucellosis and age between 15 to 75 years. Conclusions: The developed microtiter assay is highly sensitive and specific, with minimal time of reaction. Moreover; this assay is rapid, easy to apply, cost effective, and requires no laboratory set ups for its application and can be deployed for on field testing of differential diseased cases. was concluded that the solution to the problems of epidemiological investigation of Brucella outbreaks to ensure precise diagnosis will require employing a microtiter test possessing different tasks of the immune response. Predictive values were found to be appropriate high sensitivity, specificity during screening. Therefore; anew diagnosis by microtiter is very reliable and accurate diagnosis of brucellosis.