Abstract: The root system, as one of the primary organs of plants, often directly or indirectly suffers from osmotic stresses, such as drought, salinity, extreme temperatures, heavy metals, etc., which cause irreversible damage to plants. Regulating root growth is one of the most effective strategies to improve the aboveground survival quality and stress resistance of plants. Exogenous hormones have a particularly significant regulatory effects on root growth, and melatonin (MT), as an emerging plant growth regulator, has attracted much attention in stress response research. This article reviews the biosynthesis process of melatonin (which is derived from tryptophan and is produced through multiple enzymatic reactions), the main synthesis sites (chloroplasts and mitochondria), its distribution (seed, leaf, and the endodermis, pericycle, and root apical meristem zone of the root system, etc.), and its close relationship with hormones such as auxin, ethylene, etc. Under osmotic stress, MT promotes seed germination and hypocotyl development, regulates the growth of main root and lateral roots (low concentrations MT stimulate primary root elongation, while high concentration MT promote lateral root formation), and improves root morphological characteristics (increasing the number of root hairs, surface area, biomass, and root activity, etc.). This process, on the one hand, affects root growth and development through the interaction between MT and hormones and the regulation of related gene expression; on the other hand, it activates the antioxidant system, regulates the expression of stress resistance genes, and enhances the activities of superoxide dismutase, catalase, etc. to remove toxic substances such as hydroxyl groups, singlet oxygen, hydrogen peroxide and reactive oxygen species, etc. In addition, MT also affects plant root growth by influencing the structure of the rhizosphere microbial community, promoting the colonization of beneficial bacteria, improving soil nutrient utilization, and improving the root environment. In summary, melatonin's stress resistance has broad-spectrum properties, its effect is regulated by concentration, and there are differences in responses between monocotyledons and dicotyledons. Its core mechanism includes activating the antioxidant system to promote water absorption by the root system, coordinating the regulation of metabolic pathways to enhance the stress resistance of plants, and integrating the multi-level signal transduction network across stressors to form a rapid perception-balance system. This review clarifies the unique value of melatonin in coordinating "root system configuration optimization - stress resistance enhancement", providing important theoretical references for further using melatonin-mediated root system configuration regulation and rhizosphere microenvironment improvement, and also indicating the direction of using biotechnology to regulate the endogenous melatonin level of plants to enhance stress resistance.