In order to establish a rapid，sensitive and specific method for detection of Toxoplasmas gondii（T. gondi），a pair of specific primers and FAM（fluorescein）-labeled MGB probes were designed based on the conserved gene sequences of T. gondii. Followed by optimizing PCR reaction system and conditions，a real-time fluorescent PCR assay was established，and the specificity，sensitivity and reproducibility test were carried out；then 60 clinical samples suspected of T. gondii infection were tested by the established method. The results showed that the method expressed a good linear relationship when the template was within the range of 101–107 copies/μL. Specificity test showed that positive signal was observed only when amplifying the recombinant plasmids of T. gondii，rather than the water of negative contrast or other 7 pathogens；the minimum concentration of detection template was 10 copies/μL；32 out of 60 suspected samples were detected positive，and the result was consistent with that of cloning and sequencing. As a conclusion，the established method in this study could be used for rapid detection of T. gondii，which provided a specific，sensitive and high-throughput method for the identification of toxoplasmosis.