Tobacco is an excellent model plant to study developmental processes and cellular biology. Surface dielectric barrier discharge (sDBD) was used to generate plasma activated water (PAW), and the PAW was indirectly applied to study tobacco plant growth during the early developmental stages. Solid media were treated with the PAW for 8- and 18-day-old seedlings, and the effects on both the vegetative and root phenotypes were observed during the early developmental stages. In the vegetative tissue, the cotyledon size was large for the seedling that was cultivated by plasma activated water for the 12 min PAW (PAW12) treatment. The PAW dramatically elongated the cotyledon by 40% due to increased cell expansion in the leaf-length direction, not cell proliferation. In addition to the vegetative tissue, the root length due to the PAW was significantly enhanced at the primary root with elongated root hairs. According to molecular analysis, the NT_ COBRA-LIKE 9 (COBL) and two NT_ XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) genes, NT_XTH9 and NT_XTH15, were significantly over-expressed by the PAW treatment compared with untreated deionized water (DW), indicating that the PAW effects are from modulating the expression of target genes. These results demonstrate that PAW might promote tobacco plant growth both in the cotyledon and root hair tissues via the NT_COBL, NT_XTH9 and NT_XTH15 genes.