Trueperella pyogenes is an important opportunistic pathogen. Pyolysin (PLO) makes important contributions to the pathogenicity of T. pyogenes. However， the structure and function of PLO has not been well documented. In the current study， epitopes in domain 1-3 of PLO have been mapped using rabbit anti-recombinant PLO (rPLO) polyclonal antibodies， and then the results were re-checked by using mouse and chicken anti-rPLO polyclonal antibodies， respectively. The results indicated that the region of aa 281-393 in PLO could not elicit antibodies against linear epitopes. A total of six B cell linear epitopes have been found in domain 1 of PLO. Two of the six epitopes (EP1 and EP2) were used to immunize mice and chicken. Chicken anti-EP1 and anti-EP2 serum and mouse anti-EP2 serum could react with rPLO and corresponding epitope polypeptide in western blot assay; however， only mouse anti-EP2 serum shows weak anti-hemolysis effect in the rPLO and sheep red blood system. Our results provide some new information to the research field of PLO structure and function.