Abstract: In order to establish a convenient and efficient method for detecting Mannheimia haemolytica, the experiment transformed the Mannheimia haemolytica recombinant plasmid pET-32 a-PlpE into BL21（DE3） competent cells which was then induced for expression by IPTG and purified. Recombinant protein PlpE was used as the coating antigen and the test conditions was optimized such as the best coating concentration, the best dilution of the primary antibody and the secondary antibody, the best reaction time, and the best color development time; an indirect ELISA method for detecting PlpE antibody was established. The results showed that the recombinant plasmid pET-32 a-PlpE was transformed into competent cells BL21（DE3） and induced by IPTG, and the recombinant protein was mainly expressed in a large amount in the precipitate; after purification by the inclusion body purification kit, a higher purity target protein was obtained. The recombinant protein PlpE had good reactivity, and the size of the protein met expectations. The determined detection conditions were as follows: antigen coating concentration was 2 μg/mL, primary antibody dilution was 1∶300, enzyme-labeled secondary antibody dilution was 1∶80 000, enzyme-labeled secondary antibody reaction time was 45 min, primary antibody reaction time was 30 min, and the color development time was 15 min. Using this method, the lowest titer for detecting Mannheimia haemolytica positive serum could reach 1∶1 280. Using this method to detect Bovine viral diarrhea virus positive serum, Bovine alphaherpesvirus positive serum, Foot-and-mouth disease virus positive serum, Mycobacterium avium paratuberculosis positive serum, Brucella positive serum, the results were all negative. The coefficients of variation of intra-assay repeatability test and inter-assay repeatability test were 0.70%-4.70% and 0.50%-5.60%, respectively, which were both less than 6.00%. The method had good repeatability within and between batches. The results suggested that the indirect ELISA method for detecting PlpE antibodies established in this experiment had high sensitivity and specificity, and could be used for clinical detection of antibodies in bovine serum.