In recent years, gene transfer to cereal crops has become an important tool for improving agronomic traits. Barley is one of the most important cereals and like other crops its modification by genetic transformation is an important method for increasing tolerance to biotic and abiotic stresses. Optimization of the various factors that can influence transformation and regeneration of the target plant is the first step in the gene transfer. In this research, some of the factors influencing transformation of barley with Agrobacterium such as the Agrobacterium strain, bacterial concentration, co-cultivation period and Agrobacterium infection medium were investigated. To confirm the presence of possible transferred genes in the transgenic plants, a PCR test was done and expression of the transferred gene was carried out using a GFP gene expression test. Regeneration of transgenic explants was carried out successfully and fertile transgenic plants were obtained. The results showed that in the transformation of immature embryo explants of barley, the LBA4404 and AGL1 Agrobacterium strains were the most effective strain and a concentration of OD600=1 was the optimal bacterial concentration. Also, the usage of LB as the inoculation medium and a 2-days co-cultivation period of explants with Agrobacterium resulted in maximum transformation of barley.