Low-temperature stress is an important limiting factor to tobacco growth in early spring of south China. In this study, a low-temperature-resistant agent (LTRA) was employed to examine its ameliorating effect on the inhibition of tobacco growth triggered by low-temperature stress. Results indicated that low-temperature stress of 12 °C for 6 days reduced root number and biomass of tobacco seedling by 27.4% and 24.1%, while treatment with LTRA could recover the inhibitory effect of low-temperature stress on tobacco growth significantly. The content of ascorbic acid and the activities of superoxide dismutase and catalase at low-temperature stress were 65.2%, 53.5% and 32.1% of those at normal temperature condition (26 °C), while the corresponding values with LTRA treatment were 89.2%, 88.9% and 74.2%, suggesting that LTRA treatment could enhance the activity of antioxidant enzyme and the synthesis of antioxidant compounds. Low-temperature stress increased the membrane permeability by 84.8%, while LTRA treatment recovered it by 77.4%. Furthermore, LTRA treatment contributed to increase chlorophyll synthesis and maintain the integrity of tobacco leaf structure. Effective component analysis indicated that the complex of ammonium calcium nitrate and glycine betaine was the main effective component of LTRA in maintaining membrane integrity. Its effective concentration was 1.0 g L−1. The above results suggested that LTRA could enhance the synthesis of chlorophyll, activate the activity of antioxidant enzyme, maintain the integrity of cell membrane, and thus elevate the tolerance of tobacco seedlings to low-temperature stress.