Toxoplasma gondii contain various immunogenic antigens. The most important Toxoplasma antigens are somatic and excreted/secreted antigens. Rhoptry proteins are known as excreted/secreted antigens. These antigens have been proposed as a vaccine candidate against toxoplasmosis. The main objective of the present work was cloning rhoptry protein1 (ROP1) Gene of Toxoplasma gondii (RH) in a cloning vector for gene analysis and further production of rhoptry proteins. Tachyzoites of the RH strain of T. gondii were harvested from the peritoneal fluid of mice that has been experimentally infected with the parasites. Genomic DNA was extracted by phenol- chloroform method. The ROP1 fragment amplified with specific primers. The purified PCR products were ligated between the EcoR1 and BamH1 sites of the pTZ57R/T cloning vector and transformed into Escherichia coli TG1 strain and screened by IPTG and X-Gal. The plasmid was purified and visualized under UV transilluminator. The amplified fragment was cloned in pTZ57R vector successfully. The correct orientation of the ROP1 fragment was identified by restriction enzyme analysis and sequencing of constructed plasmid. A fragment about 760bp was separated from PTZ57R following digestion and demonstrated on agarose gel electrophoresis.The sequence of this amplified gene showed homology up to 96% with target gene in GenBank database (Accession no. M71274). Recombinant plasmid of ROP1 gene was constructed. It is ready for future study.