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Background: Low temperatures disrupt nitrogen metabolism in tobacco, resulting in lower nicotine content in the leaves. 24-epibrassinolide (EBR) is a widely used plant growth regulator known for its roles in enhancing cold tolerance and nitrogen metabolism. Nevertheless, it remains unclear whether EBR enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco. Results: To investigate the effects of EBR on leaf nicotine content under low temperature conditions during the mature stage of 'Yunyan 87' flue-cured tobacco, four treatments (foliar spraying of 0, 0.1, 0.2 and 0.4 mg·L− 1 EBR solutions) were performed by using a single-factor randomized complete block design. The result showed that foliar spraying of different concentrations of EBR notably improve the agronomic and economic traits of flue-cured tobacco to varying degrees, as well as increase the total nitrogen and nicotine content in the tobacco leaves. 0.2 mg·L− 1 EBR treatment showed better results, with nicotine content in the middle and upper leaves after curing increasing by 11.11% and 19.90%, respectively, compared to CK. Compared to the single EBR, foliar spraying of EBR compound containing α-Cyclodextrin and Tween 80 prolongs the effect of EBR, promotes the growth and development of tobacco plants. Combining EBR with CaCl2 and ZnSO4·7H2O significantly enhances the cold resistance of tobacco plants. Furthermore, combining EBR with higher concentrations of KH2PO4 is more effective in promoting the maturation and yellowing of the upper leaves than those with lower concentrations. Conclusions: This study provides new insights that foliar application of EBR enhances leaf nicotine content under low temperature conditions during the mature stage of flue-cured tobacco by regulating cold stress tolerance. The integration of EBR with α-Cyclodextrin, Tween 80, CaCl2, ZnSO4·7H2O and KH2PO4 showcases a novel approach to extending the effectiveness of plant growth regulators and improving agricultural sustainability. Furthermore, these findings may be applicable to other cold-sensitive crops, offering broader benefits for improving resilience and productivity under low temperatures. However, the research focuses on two growth cycles, without investigating the long-term impact of EBR on soil health, crop sustainability, and ecosystem. And further research is needed to elucidate the molecular mechanisms of EBR on enhancing leaf nicotine content. Clinical trial number: Not applicable. [ABSTRACT FROM AUTHOR]