The present study aims to investigate the impact of externally applied stevioside (a sugar-based glycoside) on soybean root growth by examining morpho-physiological characteristics, biochemical parameters, and gene expression. Soybean seedlings (10-day-old) were treated with stevioside (0, 8.0 & mu;M, 24.5 & mu;M, and 40.5 & mu;M) for four times at six days' intervals by soil drenching. Treatment with 24.5 & mu;M stevioside significantly increased root length (29.18 cm plant-1), root numbers (38.5 plant-1), root biomass (0.95 g plant-1 FW; 0.18 g plant-1 DW), shoot length (30.96 cm plant-1), and shoot biomass (2.14 g plant-1 FW; 0.36 g plant-1 DW) compared to the control. Moreover, 24.5 & mu;M of stevioside was effective in enhancing photosynthetic pigments, leaf relative water content, and antioxidant enzymes compared to control. Conversely, plants exposed to a higher concentration of stevioside (40.5 & mu;M), elevated total polyphenolic content, total flavonoid content, DPPH activity, total soluble sugars, reducing sugars, and proline content. Furthermore, gene expression of root growth development-related genes such as GmYUC2a, GmAUX2, GmPIN1A, GmABI5, GmPIF, GmSLR1, and GmLBD14 in stevioside-treated soybean plants were evaluated. Stevioside (8.0 & mu;M) showed significant expression of GmPIN1A, whereas, 40.5 & mu;M of stevioside enhanced GmABI5 expression. In contrast, most of the root growth development genes such as GmYUC2a, GmAUX2, GmPIF, GmSLR1, and GmLBD14, were highly expressed at 24.5 & mu;M of stevioside treatment. Taken together, our results demonstrate the potential of stevioside in improving morpho-physiological traits, biochemical status, and the expression of root development genes in soybean. Hence, stevioside could be used as a supplement to enhance plant performance.