An indirect immunofluorescence assay(IFA)was set up to detect Trueperella pyogenes using the rabbit hyperimmune serum. Specific conditions including fixing strategy, blocking solution, washing solution, antibody dilution, and antibody incubation time had been tested. The results showed that cold acetone as the fixative, 10% goat serum as the blocking solution, and 0.05% Tween-20 of 0.01 mol/L pH7.4 PBS as the washing solution were optimal for the method. And, the rabbit anti-Trueperella pyogenes hyperimmune serum with dilution 1∶50, incubation for 2 h at 37 °C, and secondary antibody with dilution 1∶100, incubation for 2 h at 37 °C, by use of ammonium oxalate crystal violet as stain for 3 min, the specific fluorescence was clearly visible in the pure cultivation of Trueperella pyogenes. It can clearly distinguish Arcanobacterium hemolysis, Sheep type Corynebacterium pseudotuberculosis, Cp5 type Staphylococcus aureus, O8 type E. coli, Enteritis type Salmonella, Streptococcus suis type 1. To verify the method, in dead mouse tissues infected Trueperella pyogenes including heart, liver, spleen, lungs and kidneys, and diseased goats clinical sample, Trueperella pyogenes were detected.