The northern highbush blueberry variety 'Duke' was used as the test material, and different concentrations of 2,4-Epibrassinolide (EBR) (0, 0.2, 0.4, 0.6, and 0.8 mg·L-1) were applied during the bud expansion stage, with a second application administered at one-day intervals following the first. Samples were collected at the bud, flower, and fruit stages and subsequently treated with artificial low temperatures (2°C) after sampling. The effects of various concentrations of exogenous EBR on the physiological indices of cold resistance and the expression of the cold resistance gene VcCBF3 in blueberry buds, flowers, and young fruits were investigated through comprehensive evaluation and correlation analysis. The objective was to identify the optimal concentration of EBR to enhance the cold resistance of blueberries. The results indicate that: (1) Under low temperature stress, the contents of soluble sugar, soluble protein and proline increased, along with the activities of superoxide dismutase, peroxidase, and catalase. The expression of the VcCBF3 gene expression and the ascorbate-glutathione cycling system were up-regulated, and with the increase of EBR concentration, the expression of the VcCBF3 gene initially rose and then declined. The content of malondialdehyde and the production rate of superoxide anion radicals decreased, and with the increase of EBR concentration, the content of malondialdehyde first decreased and then increased. (2) Overall low temperature resistance, flowers >
buds >
young fruits. (3) Appropriate concentrations of exogenous EBR can effectively mitigate freezing damage in blueberries caused by low temperatures. A comprehensive evaluation and correlation analysis of each cold tolerance index and the expression of the VcCBF3 gene revealed that a treatment concentration of 0.4 mg·L-1 had the most significant mitigating effect among the sprayed EBR concentrations of 0, 0.2, 0.4, 0.6, and 0.8 mg·L-1.