Objective Triosephosphate isomerase (TPI) of Toxoplasma gondii is an immunodominant antigen secreted by T. gondii during infection and therefore an important diagnostic antigen. To explore its potential for diagnostic purpose, the polyclonal antibody anti-TPI was made in rabbit and its application to detect T. gondii infection was explored. Methods A New Zealand white rabbit was immunized with 200 μg recombinant TPI protein expressed in Escherichia coli and emulsified with Freund’s adjuvant for three times. The serum was collected from the immunized rabbit 2 weeks after the last immunization. The total IgG in immunized-rabbit serum was purified with Protein A affinity purification column. SDS-PAGE was used to detect the purity of the purified IgG. The anti-TPI antibody titers in the purified IgG and in the immune sera was measured by ELISA and its specific recognition of recombinant TPI and native TPI in T. gondii excretory/secretory (ES) products was detected by Western blotting. The anti-TPI IgG was conjugated on magnet beats to pull down the TPI in T. gondii-infected mouse sera and the pull-downed TPI was applied on NC membrane to be detected by the anti-TPI IgG, and the results were compared with that detected by ELISA kit. Results The recombinant TPI protein was expressed in E. coli and the purified TPI protein was used to immunize a rabbit. The rabbit anti-TPI serum was obtained and the total IgG in the serum was purified with Protein-A affinity column. SDS-PAGE analysis identified that the purified IgG contained typical IgG heavy chain and light chain. The anti-TPI specific antibody titers were 1 : 128 000 in immunized rabbit serum and in the purified IgG measured by ELISA. Western blotting showed that the recombinant TPI protein and native TPI in T. gondii ES products could be specifically recognized by the purified IgG. The purified rabbit IgG was able to pull down the TPI antigen in 10 of 11 sera of mice infected with T. gondii, however, one of the 11 normal mouse sera also showed positive. The results detected by ELISA kit showed that 6 of the 11 infected mice were positive and 5 were negative; 11 healthy mice were all negative. There was no significant difference in the results between Dot blot and ELISA detection. Conclusion The anti-TPI polyclonal antibody was prepared in immunized rabbit. An anti-TPI IgG-based immunological assay was potentially established to detect T. gondii infection. This TPI-specific IgG could be used to develop an immunodiagnostic assay to detect T. gondii infection.