Elicitors could be used as the enhancer of plant secondary-metabolite synthesis and could play an important role in biosynthetic pathways of important medicinal compounds. The present study investigated the effect of abiotic elicitor (fungal extract of Fusarium graminearum) and two abiotic elicitors (methyl jasmonate and salicylic acid) at different concentrations in 18- and 30-day-old cultures of hairy roots infected with two strains of Agrobacterium rhizogenes, A13 and 9534. The hairy roots were harvested 48 and 96 h after inoculation. Polymerase chain reaction analysis (PCR) with specific primers rolB and virD genes was performed to confirm the transgenic hairy roots production. Detection and identification of lignan was carried out by high performance liquid chromatography (HPLC) method. The results of PCR analysis showed diagnostic bands 780 bp in size related to specific reproduction of rolB gene. Also maximum lignan production for each elicitor was as follow: fungal extract at 1% v/v (12.87±0.66 and 89.65±3.9 mg/gr DW±SD podophyllotoxin and 6-methoxypodophyllotoxin, respectively for), methyl jasmonat at 100 mm (11.37±0.65 and 75.65±3.9 mg/gr DW±SD for podophyllotoxin and 6-methoxypodophyllotoxin, respectively), and salicylic acid at 200 mm (7.97±0.33 and 51.68±2.1 mg/gr DW±SD for podophyllotoxin and 6-methoxypodophyllotoxin, respectively). Important factors such as strain of agrobacterium (A13), duration of exposure time (48 h), and age of culture (18-day-old culture) affected lignan accumulation.