Humic substances（HS）are one of the most stable forms of organic matter with in vitro biological activity. This study reviews the chemical composition, structural characteristics, and physiological activity of HS and discusses the internal mechanism of the physiological activity based on the relationship between these aspects. The chemical composition of HS is uncertain, and the supramolecular structure model of HS is currently generally accepted. HS promotes the absorption and utilization of mineral elements by promoting the formation of root hairs and lateral root development, activating plasma membrane H⁺-ATPase to generate favorable electrochemical gradients and chelating mineral elements with acidic functional groups. With hormone-like activity, HS IAA secreted by plant rhizosphere growth-promoting bacteria（PGPR）or HS autologous component can activate plasma membrane H⁺-ATPase. HS can also increase endogenous plant hormone levels and promote plant growth and development. HS activates plasma membrane H⁺-ATPase by mediating messenger NO to eliminate reactive oxygen species（ROS）generated from HS-affected cells. HS mediates root development and morphological structure by regulating intracellular ROS homeostasis through NO and antioxidant enzymes. This study additionally focuses on the correlation between HS physiological activity and its chemical composition; the role of active sites such as —COOH and —OH, the electron transfers of quinone groups, and HS hydrophilic–hydrophobic balance are closely related to HS physiological activities. Future HS research should focus on analyzing HS molecular structures, clarifying the“common structural element”with physiological activity of the cross-feedback mechanism between HS-induced intracellular signal transduction pathways, and revealing the regulation mechanism of HS on gene expression related to material and energy metabolism in cells, aiming to provide a theoretical basis for the in-depth study of HS physiological activity mechanism and agricultural applications.