Chemical synthesis issue of tropane alkaloids and their lowest production rate along with their extraction costs has made use of new methods necessary to produce valuable pharmaceutical compounds. hairy roots induction end elicitation with biotic or abiotic elicitors such as nanoparticles can be used as a source for high production of worth metabolites compared to intact plants. Hyoscyamus reticulatus L. is a rich source of hyoscyamine and scopolamine, which are widely used in pharmaceuticals. Since the in this research, the effect of different concentrations (0, 25, 50, 75 and 100 mg/l) of zinc Nano oxide (ZnO NPs) as a stimulant on the growth rate, antioxidant enzyme activity and antioxidant activity, total phenol content (TPC), total flavonoid content (TFC) and tropane alkaloids in the H. reticulatus hairy roots was investigated in a bubble column bioreactor. The highest and the lowest amount of fresh weight (FW) (153.4 and 79.82 g) were recorded in 50 mg/l during 48 h exposure time and 100 mg/l during 72 h respectively. Also, ANOVA results showed that the interaction effect of concentration and exposure time was significant on guaiacol peroxidase (GPX) enzyme activity, TPC and TFC, antioxidant activity and alkaloid content (p <
0.01). HPLC analysis revealed that the highest amount of hyoscyamine and scopolamine (400 and 275.1 mg/kg of DW) were observed at ZnO NPs (75 mg/l) during 72 h respectively. According to the results, it can be said that the use of nano zinc oxide as a stimulant is an effective method for increasing tropane alkaloids in a bubble column bioreactor.