Hairy root induction by Agrobacterium rhizogenes is a reliable method to increase P. oleracea medicinal compounds. The hairy roots in addition to high growth rates are genetically and biochemically stable. Purslane (Portulaca oleracea L.) is one of the valuable medicinal plants and has important secondary metabolites such as dopamine and noradrenaline. In this study, hairy roots of Purslane were produced by using Agrobacterium rhizogenes strain A₄ and different concentrations of macro elements (0.5x, 1x, 1.5x, 2x and 2.5x concentrations of MS base medium) were investigated. For confirming hairy roots transformation, the presence of the rolB gene in the hairy roots was performed by PCR and using rolB gene's specific primers. The results show that maximum hairy root induction was observed by using 0.5x KNO₃ (80 percentage). The highest phenolic content was obtained by using 0.5x KNO₃ (4.52 mg/g DW) and also maximum flavonoid content, (18.2 and 17.02 mg/g DW respectively) was belonged to 0.5x and 1x KNO₃. Molecular confirmation of transgenic hairy roots was done with PCR reaction.