The invention relates to a recombinant Wzt protein rabbit serum polyclonal antibody and a preparation method thereof, which relate to the field of antibody preparation, and fill the gap of applying recombinant Wzt protein for preparing the specific polyclonal antibody. The preparation method of the polyclonal antibody comprises the steps of after mixing and emulsifying the recombinant Wzt protein and an equivoluminal freund's complete adjuvant, carrying out multiple sites subcutaneous injection on the back parts of female rabbits; carrying out secondary immunizing: after one week, after mixing and emulsifying the recombinant Wzt protein and the equivoluminal freund's incomplete adjuvant, carrying out multiple sites subcutaneous injection on the back parts of the female rabbits; carrying out thrice immunizing and quartic immunizing every other week according to a secondary immunizing process; collecting venous blood of female domestic rabbit ears after immunizing each time, and detecting a rabbit serum antibody titer; at one week after quartic immunizing, collecting rabbit whole blood, centrifugally collecting a supernatant to obtain a rabbit serum, and obtaining the recombinant Wzt protein rabbit serum polyclonal antibody after purifying. The recombinant Wzt protein rabbit serum polyclonal antibody has favorable specificity, and can be used for detecting Brucella Wzt protein.