Ascorbic acid (AsA), an essential non-enzymatic antioxidant in plants, regulates development growth and responses to abiotic and biotic stresses. However, research on AsA's role in cold tolerance remains largely unknown. Here, our study uncovered the positive role of AsA in improving cold stress tolerance in tomato seedlings. Physiological analysis showed that AsA significantly enhanced the enzyme activity of the antioxidant defense system in tomato seedling leaves and increased the contents of proline, sugar, abscisic acid (ABA), and endogenous AsA. In addition, we found that AsA is able to protect the photosynthetic system of tomato seedlings, thereby relieving the declining rate of chlorophyll fluorescence parameters. qRT-PCR analysis indicated that AsA significantly increased the expression of genes encoding antioxidant enzymes and involved in AsA synthesis, ABA biosynthesis/signal transduction, and low-temperature responses in tomato. In conclusion, the application of exogenous AsA enhances cold stress tolerance in tomato seedlings through various molecular and physiological responses. This provides a theoretical foundation for exploring the regulatory mechanisms underlying cold tolerance in tomato and offers practical guidance for enhancing cold tolerance in tomato cultivation.