The level of root development is a crucial factor determining the formation of vigorous flue-cured tobacco seedlings and their subsequent growth potential after transplanting. However, issues such as insufficient root development and limited nutrient absorption capacity remain prominent during the float breeding process, highlighting the urgent need to explore safe and efficient ecological root-promoting measures. Based on the combined regulatory potential of functional active substances, a compound biostimulant was formulated using carboxymethyl chitosan, γ-aminobutyric acid, and γ-polyglutamic acid. A hydroponic experiment with different application timings and concentration gradients was conducted to reveal the dose-response effects and regulatory pathways of this compound biostimulant on the root architecture and physiological metabolism of tobacco seedlings. The results demonstrated that the compound biostimulant exhibited a significant biphasic dose-response characteristic (hormesis) on seedling growth, characterized by stimulation at low doses and inhibition at high doses, and this response was highly dependent on the application timing. Within the tested range, the low-concentration treatment applied at the early seedling stage (L) exhibited the optimal growth-promoting effect, significantly increasing the shoot and root dry weights by 42.0% and 82.8%, respectively, compared with the control (CK). Morphological analysis indicated that the low-concentration treatment optimized root architecture by promoting the formation of lateral and fine roots, leading to significant increases of 97.0% and 125% in total root surface area and root tip number, respectively. Physiologically, the low-concentration treatment significantly enhanced root activity by 2.92 fold compared to the CK, and concurrently increased leaf SPAD values and root nitrate reductase (NR) activity. Conversely, high-concentration treatments (H, H30) inhibited seedling growth and were accompanied by an abnormal accumulation of root soluble proteins, implying that excessive application might trigger osmotic or physiological stress responses. Correlation analysis further confirmed that the optimization of root architecture was significantly and positively correlated with seedling biomass accumulation and carbon-nitrogen metabolism indicators. This study elucidates the physiological response pattern of the compound biostimulant, demonstrating that moderate stimulation promotes growth whereas excessive application induces stress, thereby providing a theoretical basis for the scientific application of multi-component ecological root-promoting agents in tobacco float breeding.