In order to develop an accurate and rapid method for the detection of Mycoplasma haemocanis, an indirect ELISA method based on phosphoglycerate kinase (PGK) of Mycoplasma haemocanis was established. The phosphoglycerate kinase (PGK) gene sequence of Mycoplasma haemocanis was obtained from the published whole genome sequence of Mycoplasma haemocanis in NCBI. By selecting the codon optimized gene sequence preferred by prokaryotes and inserting the chemically synthesized new gene sequence pgk into the plasmid, the prokaryotic expression vector pet32a(+)-pgk was successfully constructed and expressed in E. coli BL21. The relative molecular weight of the protein was confirmed to be about 60 ku by SDS-PAGE. Using purified PGK recombinant protein as coating antigen, an indirect ELISA method based on PGK protein was established and further optimized. The results showed that the optimum coating concentration of antigen was 2.5 μg/mL, the optimum dilution multiple of serum was 1∶200, the optimal working concentration of sealant was 5% skim milk, the optimal sealing time of sealant was 45 min, the optimal action time of serum to be tested was 60 min, the optimal working concentration of enzyme labeled second antibody was 1∶5,000, and the optimal action time of enzyme labeled second antibody was 30min. Based on this, an indirect ELISA method for canine Mycoplasma haemocanis was established, and the clinical negative serum was selected to calculate the critical value. This method was used to detect 166 clinical samples, and the positive rate was 21.1%, which was consistent with the result of fluorescence quantitative PCR. The completion of this experiment provides an effective detection method for clinical monitoring of Mycoplasma haemocanis in dogs and provides a research basis for further prevention and treatment of the disease.