The effects of different concentrations of exogenous 2,4-epibrassinolide(EBR) and 5-aminoacetyl propane (ALA) on the growth, antioxidant enzyme activity, osmoregulatory substance content and nitrogen metabolism key enzyme activity of overwintering pepper seedlings were studied using ‘Bola Red Bull’ ‘Xingshu 215’ and ‘Changyan Qingxiang’ as test materials. The results showed that exogenous EBR and ALA could enhance the ability of seedlings to resist low temperature stress to varying degrees. Compared to the water-sprayed control, the application of different concentrations of exogenous EBR and ALA improved the morphological indicators of overwintering pepper seedlings, including plant height, stem thickness, fresh weight, dry weight, and seedling vigor index. The activities of antioxidant enzymes(SOD, POD, CAT) were enhanced, and the content of osmoregulatory substances(proline, soluble sugars, soluble proteins) and the activities of key nitrogen metabolism enzymes(NR, NiR, GS) were also increased to some extent. Among them, 0.01 mg/L EBR and 30.00 mg/L ALA were more effective in alleviating the damage effects of low-temperature stress on seedling physiology and growth. Based on these findings, practical application trial was conducted for overwintering pepper seedling cultivation at this concentration. The results showed that the seedling vigor index following the foliar application of 30.00 mg/L ALA plus an adjuvant was significantly higher than that in all other treatments, and the additional cost per seedling was calculated to be RMB 0.007 3.