Background and aim: Leishmaniasis is a group of diseases with various clinical pictures, which is caused by Leishmania spp. This parasite causes disease in human and about one hundred animal species. The disease is widely distributed in Iran and also across the world. One of the best approaches in the development of vaccine against Leishmania is genetically modification of the parasite. Therefore, the aim of this study was to design a novel genetic construct in order to insert Herpes Simplex Virus thimidine kinase (HSV-tk) and YEAST cytosine deaminase (YEAST-cd) suicide genes into the Leishmania genome.Methods: In this work, at the first step, HSV-tk and YEAST-cd fragments along with a gene of Leishmani, a-tubuline were cloned into pBluescript vector and the arrangement of tk-atub-cd was created. Subsequently, these fragments were cut off from the plasmid and sub cloned into the plasmid pF4X1.4.4sat. The final construct was confirmed by digestion with relevant restriction enzymes. Results: The fragments tk- atub-cd was cloned successfully in the plasmid pBluescript and its authenticity was confirmed. Subsequently, this genetic collection was inserted into the plasmid pF4X1.4sat and finally, the orientation of it was checked.Conclusion: The construct designed in this study was able to insert two cellular suicide genes HSV-tk and YEAST-cd into the Leishmanai genome. Thus, this is an approach in achievement of vaccine against Leishmanai in the coming up researches.