<p>Quantum dots are promising nano-elicitors that modulate plant physiology via redox signaling and metabolic adjustments. This study explores their effects on medicinal plant tissue cultures, specifically focusing on <i>Calotropis procera</i>. Therefore, this study aimed to establish an optimized protocol for callus induction in <i>C. procera</i> and evaluate the dose-dependent physiological and biochemical responses of callus to copper quantum dots (Cu-QDs). Callus cultures were successfully initiated from leaf explants on MS medium supplemented with naphthalene acetic acid (0.5 mg L^− 1), kinetin (1.0 mg L^− 1), and 2,4-dichlorophenoxyacetic acid (0.2 mg L^− 1), producing semi-compact, friable calli with a high proliferation capacity. Subsequently, we exposed the calli to varying concentrations of Cu-QDs (ranging from 0 to 100 mg L^− 1) to assess their physiological and biochemical responses. A hormetic response was observed in this study. At 5 mg L^− 1, Cu-QDs enhanced biomass production, protein content, and antioxidant enzyme activity, while reducing malondialdehyde levels. Specialized metabolite levels, including phenolics (27%), flavonoids (34.7%), and anthocyanins (131.5%), were markedly elevated compared to the control. GC-MS analysis revealed increased accumulation of volatiles at 5 mg L^− 1, including sabinene (~ 250%), β-ocimene (~ 110%), linalool (~ 26%), myristicin (~ 29%), and methyl eugenol (~ 50%). Conversely, higher concentrations (≥ 75 mg L^− 1) suppressed growth and triggered oxidative stress, as evidenced by the upregulation of superoxide dismutase and ascorbate peroxidase, along with a marked decline in catalase activity. These results demonstrate that Cu-QDs, when applied at optimal doses, act as efficient elicitors that modulate metabolic and redox responses in <i>C. procera</i> calli, providing a novel approach to boost phytochemical yields in vitro cultures.</p>