DNA-binding one zinc finger (DOF) proteins play a key role in plant resistance to drought conditions. In this paper, a DOF gene (VyDOF8) identified from Chinese wild grapevine Vitis yeshanensis 'Yanshan', was significantly increased by salt, drought, and cold stresses. The VyDOF8 protein was localized to the nucleus and functioned as a transcription factor. VyDOF8-overexpressing (VyDOF8-OE) tobacco (Nicotiana benthamiana) exhibited a smaller percent water loss and advanced root development, and accumulated more chlorophyll and proline under drought conditions compared with the control, which suggested that overexpression of VyDOF8 might confer enhanced drought tolerance by increasing the abscisic acid content and promoting stress-responsive gene expression. Meanwhile, VyDOF8-OE plants also showed reduced malondialdehyde (MDA) and H₂O₂ contents by boosting key enzymatic antioxidant activities under drought conditions. These findings provide valuable information for functional analysis of DOF genes in grape.