To address the problem of frequent late spring coldness in tobacco-growing areas of northern Guangdong, which hinders the growth of tobacco seedlings, this study aimed to clarify the low-temperature adaptability and physiological response characteristics of the main tobacco cultivar ‘Yueyan 97’ to support cold-resistant management during the seedling stage. The tobacco seedlings at the 4-leaf, 6-leaf, and 8-leaf stages were used as materials, and the low-temperature treatments of 5, 10, and 15℃ and the control treatment of 25℃ were set up in this study. After 24 hours of cultivation, the leaf chlorophyll content, nitrogen content, superoxide dismutase (SOD) activity, peroxidase (POD) activity, and soluble protein content were determined for analyzing the characteristics of low-temperature adaptability. The results showed: (1) The response of antioxidant system and osmotic adjustment substances were significantly induced by low temperature, and the SOD activity, POD activity and soluble protein content at 5℃ were increased by 32%-54%, 44%-54% and 23%-60%, respectively, compared with the control at 25℃. The chlorophyll and nitrogen content reached the highest at the 4-leaf stage, reaching 52.62 SPAD and 18.08 mg/g at 5℃, respectively, which increased by 11%-168% compared with other seedling ages. (2) The difference of seedling age significantly affected the adaptability to low temperature. The chlorophyll and nitrogen content were dominant in the 4-leaf stage, while SOD activity [53.93 U/(g·FW)], POD activity [138.56 U/(g·FW)] and soluble protein content [28.43 mg/(g·FW)]were dominant in the 8-leaf stage, which were significantly better than those in the 6-leaf stage. (3) The correlation analysis showed that chlorophyll content was positively correlated with nitrogen content (r=0.916-0.998), SOD activity was positively correlated with soluble protein content at 5℃ (r=0.917), and POD activity was positively correlated with soluble protein content at 10℃ (r=0.741). The results showed that the seedlings of ‘Yueyan 97’ exhibited good adaptability to low-temperature conditions, the seedlings at 4-leaf stage and 8-leaf stage showed the best performance, and its low temperature adaptation mechanism was closely related to the synergistic effect of nitrogen metabolism, antioxidant system and osmotic adjustment substances. This study can provide data support for the further research on the cold resistance mechanism of ‘Yueyan 97’ and its cultivation promotion.