In the present study, hairy root production in Plumbago europaea was elicited using elicitors for the production of plumbagin, an important medicinal compound. Accordingly, factors such as elicitor concentrations and time exposure of optimal concentration of elicitors on biomass, plumbagin production, and PKS gene expression were studied. The growth of hairy roots was decreased by high concentrations of salicylic acid, methyl jasmonate, and chitosan. On the other hand, all of the elicitors increased the accumulation of plumbagin. The expression of the PKS gene was significantly increased under the influence of elicitors. The maximum amount of total plumbagin (11.66 mg/g DW) was achieved in hairy root culture treated with 150 mg/l chitosan on the third day after treatment, which was 4.6 times higher than the control roots. Adding chitosan to hairy root culture led to the increase and release of plumbagin into the media, which is highly desirable for easy recovery of the product. Chi strongly influenced plumbagin accumulation, and hence it could be suggested as an effective elicitor for the Plumbago genus. The production of plumbagin was studied using combined treatments of chitosan elicitation and in situ adsorption using XAD-7 as an adsorbent. After chitosan treatment for 48 h, the addition of XAD-7 enhanced plumbagin production which was 1.5 and 5.5-fold higher than chitosan treated and untreated hairy root culture, respectively. Accordingly, chitosan elicitation in combination with XAD-7 was a suitable approach for the production of plumbagin.