Nanotechnology is one of the advance technologies that almost found implications in every field of science. The importance is due to the unique properties of nanoparticles. In this study, bimetallic alloys of copper (Cu) and gold (Au) were tested in submerge root cultures of Stevia rebaudiana for production of biomass and secondary metabolites. A known amount of inoculum roots were submerged in liquid Murashige and Skoog medium containing combination of naphthalene acetic acid (NAA; 0.5 mg l-1) and different ratios of nanoparticles (NPs). NAA augmented medium was used as control. The addition of nanoparticles (30 µg l-1) stimulated biomass accumulation (1.447 g/flask) on 27th day of log phases. The maximum total phenolics content (TPC; 16.17 mg/g-DW) and total flavonoids content (TFC; 4.20 mg/g-DW) were displayed using AuCu-NPs (1:3) and NAA. The same combinations enhanced total phenolic production (TPP; 116 mg/L) and total flavonoid production (TFP; 29.5 mg/L) in submerged cultures. A strong correlation was observed between phenolics, flavonoids and dry biomass. Moreover, maximum 1, 1-diphenyl-2-picrylhydrazyl (DPPH) activity of 79% was displayed by addition of AuCu (1:3) nanoparticles. In conclusion, nanoparticles application has shown a positive effect in enhancing biomass and secondary metabolites production in adventitious root cultures of Stevia rebaudiana.