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