A complete P35 surface antigen of Toxoplasma gondii was sequenced (GenBank AF310261). Immunoblot found that it reacted specially with T. gondii acute infected sera, and the recombinant P35 signal was specific for P35 antigen. The P35-GST protein was used as antigen to detect 125 sera samples by double-sandwich ELISA. P35-IgM positive rate in a chronic infected group, a persistent IgM positive chronic group, a recently seroconvered group and an acute infected group were 4% (1 out of 25), 16% (4 out of 25), 88% (22 out of 25), and 100% (25 out of 25), respectively. The sensitivity and specificity of the recombinant full-length P35 antigen were 100 and 96%, respectively. The detailed expression patterns of P35 antigen were studied in 36 IgM and IgG positive sequential samples from 10 recently seroconvered patients. Results showed that the P35-IgM positive rate decreased as the time after the first seroconversion increased. P35-IgM positive samples in the first, second, third, fourth, and fifth month after the first seroconversion test were 90, 78, 57, 50, and 33%, respectively. P35-IgG positive samples in the first, second, third, fourth, fifth, sixth, and seventh month after the first seroconversion test were 70, 100, 100, 100, 67, 100, and 100%, respectively. All samples were P35-IgM negative after the fifth month, and P35-IgG negative after the seventh month from seroconversion. P35-IgM existed the shortest time and was a more specific marker for T. gondii acute infection than P35-IgG, IgM, and IgG to whole tachyzoites antigens.