Hairy root cultures due to the their higheir genetic and biological stability, high growth rate and rapid growth on hormone-free medium are an effective alternative method for production of secondary metabolites. In this research, the effects of different factors including type of Agrobacterium rhizogenes strain (A4, 15834 and 2656), type of explants (leaf and petiole), inoculation conditions (inoculation time (10 and 15 minutes), co-cultivation time (48 and 72 hours), use and non-use of asetosyringone) and salts concentration of medium (MS and 1/2MS) on induction of hairy roots in valerian were investigated. Among the Agrobacterium strains, the highest percentage of hairy root induction and the number of roots per explant was obtained using A4 strain. Also, the percentage of hairy root induction and the number of roots per explant in the leaf explant were significantly higher than those of petiole explant. Among the different inoculation conditions, the highest percentage of hairy root induction was obtained in 10 minutes inoculation and 72 hours of co-cultivation and the highest number of roots per explant was obtained in 10 minutes inoculation and 72 hours co-cultivation in medium containing 100 μM asetosyringone. Also, the percentage and number of roots per explant on ½ salt concentration of MS medium were significantly higher than those of MS medium. The HPLC results showed that valerenic acid is produced at 3.77 mg/l in V. officinalis hairy root cultures.