Abstract: 【Objective】 To establish and optimize the natural hapten agar gel immuno-diffusion test (NH-AGID) for Brucella abortus in clinical samples.【Methods】 The NH-AGID method was established to evaluate the specificity, sensitivity and reproducibility of the method; 2,287 bovine and sheep sera from immunized areas and 252 from non-immunized areas were tested. 【Results】 The NH-AGID method with stable and reproducible assay and high specificity was successfully established; The sensitivity of the assay for naturally infected positive animals was lower than 80%; The sensitivity of the assay for releasing animals was higher than 90%; The threshold for detecting bruclosis-positive antibodies was higher than RBT. The average detection rate of serum NH antibodies in immunized areas was 17.8%, and the average detection rate of LPS antibodies was 52.3%; the average detection rate of serum NH antibodies in non-immunized areas was 40.9%, and the average detection rate of LPS antibodies was 54.8%. 【Conclusion】 The NH-AGID method can be applied to identify and distinguish bovine and sheep brucellosis-infected animals from immunized animals in immunized areas. Animals detected with NH antibodies in the active or excretory phase of Brucella abortus are brucellosis-infected animals and should be removed from the herd in time.