Trachyspermum ammi is an important medicinal plant that contains a bioactive compound namely thymol. In the study, T. ammi was transformed by Agrobacterium rhizogenes strains. Seedling stem explants were inoculated with A. rhizogenes strains A4, LBA 9402, ATCC 15834, and the effect of different co-cultivation media along with incorporation of acetosyringone (100 µM) was evaluated comparatively on the frequency of hairy root induction. The polymerase chain reaction using rolB and virD specific primers was served to confirm the putative transformed hairy roots. All strains established hairy root with various frequencies, among which strain ATCC 15834 was significantly the most efficient strain for hairy root induction (84.3%). Half-strength B5 medium and incorporation of acetosiryngone (100 µM) were also significantly optimal for hairy root induction. Hairy roots culture induced by ATCC 15834 using half-strength B5 liquid medium supplemented with 30 g L sucrose indicated the highest accumulation of biomass (99.05 g L FW and 10.95 g L DW) and thymol content (11.30 mg g DM) at 20 days. Nearly 4.9-fold and 5.3-fold increment of biomass and thymol accumulation was observed, respectively, at 20 days in comparison with the untransformed control roots. The results showed the high potential of T. ammi hairy roots for the biosynthesis of thymol.