In order to Detect Toxoplasma gondii acute infection in serum samples by complete recombinant P35 surface antigen protein, JM109 cell line which can express P35-GST protein was constructed. Then the recombinant protein was separated and purified using affinity chromatography. SDS-PAGE and Western blot were used to analyse the characters of this recombinant protein. Later P35-GST protein was used as antigen to detect Toxoplasma gondii infection by IgM-ELISA. The result of SDS-PAGE showed that the recombinant protein was about 60 ku and was an hydrophile protein. It reacted specifically with Toxoplasma gondii positive serum in Western blot analysis. 60 different serum samples were detected in IgM-ELISA tests using P35-GST as antigen. It was showed that P35-GST can separate acute infection, chronic infection with IgM and IgG positive, chronic infection with IgM negative and IgG postive significantly. P35-GST was very useful in detecting acute and chronic infection of Toxoplasma gondii. It can be concluded that P35-GST can effectively separate acute and chronic infection using serum samples.