Multi-walled carbon nanotubes (MWCNTs) are cylindrical rolled sheets with different applications in plant biotechnology due to their physicochemical characteristics. This study aimed to evaluate the in vitro physiological and biochemical responses of stevia (Stevia rebaudiana Bertoni) exposure to different concentrations of MWCNTs during multiplication phase in liquid medium using temporary immersion bioreactors. The MWCNTs were characterized regarding their properties by transmission electron microscope. Different treatments (0, 25, 50 and 100 mg/L) of MWCNTs were added to Murashige and Skoog semisolid culture medium at the proliferation phase. At 30 d of in vitro multiplication, the number of shoots, shoot length, number of leaves, dry matter, and antioxidant capacity were evaluated, as well as total chlorophyll, carotenoid, macro and micronutrient, phenolic, Phosphoenolpyruvate (PEP) and Rubisco contents. The results showed that adding MWCNTs at low doses (25 and 50 mg/L MWCNT) led to an increase in the in vitro multiplication rate and a higher number of leaves, while, at the highest dose (100 mg/L MWCNT), there was an inhibition of these variables. MWCNT treatments had an effect on Mg, Fe and C elements. The accumulation of photosynthetic pigments was associated with a higher PEP content in MWCNT treatments. Phenolic content increased at a concentration of 100 mg/L MWCNT, while antioxidant capacity increased at a concentration of 50 mg/L MWCNT. In conclusion, the usage of low MWCNT doses could be used to induce a hormetic response and to promote photosynthesis during in vitro propagation of stevia.