Abstract: Objective: To establish cloning vector and standard positive control of molecular biology diagnosis of toxoplasmosis based on the 529 bp repetitive fragment to detect the presence of Toxoplasma gondii (T.gondii).Methods: The repetitive 529 bp DNA fragment from T.gondii was amplified by PCR and was inserted into pMD18-T vectors to construct recombinant plasmid and transformed to E.coli DH-5α.A pair of primers were designed and synthesized based on the 529 bp sequence to amplify the 249 bp fragment gene and plasmid DNA of T.gondii.Results: The 529 bp DNA fragment of T.gondii was successfully amplified and inserted into pMD18-T vectors after purification.The 249 bp fragment was amplified by PCR using T.gondii gemome DNA and recombinant plasmid DNA as template.Conclusions: Successfully cloned assay of 529 bp DNA fragment of T.gondii and amplified the 249 bp DNA fragment have laied the foundation of development of T.gondii diagnostic kit.