Abstract: Agrobacterium tumefaciens strain LBA 4404 harboring a binary plasmid pCambar (containing Basta resistance (bar) gene, neomycin phosphotransferase (npt II) gene and β-Glucuronidase (GUS) gene) was used to optimize the transformation efficiency in Citrullus lanatus cv. Round Dragon. In this study, the ability of Agrobacterium tumefaciens to mediate a gene transfer in Citrullus lanatus was highly dependent on various transformation factors. In current investigation, we have established factors influencing gene expression including explant ages of cotyledon- (5 days old cotyledon), pre-culture condition (2 days), wounding technique (multi wired with massive wounding technique), Agrobacterium concentration (A600 nm 0.8), co-incubation period of explants with Agrobacterium (30 min) and acetosyringone concentration (200 μM) in co-cultivation medium. These factors gave maximized transformation efficiency. The expression of the foreign functional gene in the plant genome was confirmed by histochemical GUS assay activity after 3 days of co-cultivation period. By combining the best conditions from each evaluated factors, we successfully established an efficient and reproducible Agrobacterium-mediated transformation protocol for Citrullus lanatus cv. Round Dragon which yielded 100% of transgene expression with 181.18±0.57 blue spots per responding explant. In conclusion, the transformation system for Citrullus lanatus cv. Round Dragon was optimized. The transformation procedure was proved to influence the transformation efficiency in Citrullus lanatus at 100% transient expression. Present study also suggested that different plant varieties may affect the transformation rate. The optimized conditions will then be used in future research to make a transgenic plant.