Abstract: In order to establish an indirect ELISA method with good specificity, high sensitivity and good reproducibility for the detection of blue fox Encephalitozoon cuniculi disease, in the test, firstly, the PTP2 gene was amplified from brain tissue DNA of the blue fox suspected to be infected with Encephalitozoon cuniculi, and the recombinant plasmid pET32a-PTP2 was constructed by ligating it with the prokaryotic expression vector pET-32a（+）, which was transformed into Escherichia coli BL21（DE3） competent cells for induction expression. The expressed recombinant protein was analyzed for solubility analysis after ultrasonication, and the target protein was purified. Then, using the recombinant protein as the coating antigen, an indirect ELISA method for blue fox Encephalitozoon cuniculi was established by optimizing the reaction conditions and determining the negative and positive serum cut-off values, and specificity, sensitivity and repeatability tests were carried out. Finally, the established indirect ELISA method was used to detect 28 serum samples of blue foxes infected with rabbit encephalitis microsporidiosis confirmed by conventional PCR method, and the compliance rate between this method and PCR method was calculated. The results showed that the expressed recombinant protein had a molecular mass of 45.8 ku and the expression product existed in both soluble protein and inclusion body forms; the heterobands were significantly reduced after purification, and the purified recombinant protein amount concentration was 1.55 mg/mL. The optimal mass concentration of the encapsulated antigen was 8 μg/ml; the optimal dilution of primary antibody was 1∶100; the optimal sealing solution was 5% skimmed milk; the optimal sealing conditions were 37 ℃ for 1 h; the optimal primary antibody incubation time was 120 min; the optimal dilution of enzyme-labeled secondary antibody was 1∶5 000; the optimal enzyme-labeled secondary antibody incubation time was 60 min; and the optimal color development time was 20 min. The critical values of OD₄₅₀ for negative and positive sera were 0.268 and 0.302, respectively. The established indirect ELISA method was negative for detection and blue fox canine distemper virus, blue fox pseudorabies virus, and blue fox parvovirus standard-positive sera; blue fox Encephalitozoon cuniculi-positive standard sera at a dilution of 1∶3 200 could be detected; and the coefficients of variation for intra-and inter-assay duplicates were less than 10%. The established indirect ELISA method was used to detect 23 positive samples from 28 blue fox serum samples, and the coincidence rate with the conventional PCR method was 82.14%.The results indicated that an indirect ELISA method had been successfully established for the detection of blue fox Encephalitozoon cuniculi, which had good specificity, high sensitivity and reproducibility, and was suitable for popularization at the grass-roots level.