Drought is a global problem limiting plant growth and productivity by hampering the physiological and biochemical processes. Acetylcholine (ACh), a potential neurotransmitter found in lower and higher plants, has the potential to promote growth. However, little is known about ACh-mediated physiological and biochemical changes that promote plants' growth under drought stress conditions. Current experiments were undertaken to assess the effect of ACh (0.01 and 0.1 mM ACh) supplementation on the growth performance of tobacco under drought stress (10%, PEG 6000). The current findings exhibit that drought stress substantially reduced the physiological and biochemical parameters. However, the ACh application enhanced the growth and biomass of tobacco plants. Moreover, ACh application significantly enhanced the activity of PSII and chlorophyll fluorescence under normal and drought stress treatment, respectively. Furthermore, PEG treatment increases reactive oxygen species and oxidative damage; however, ACh application reduced H2O2, O-2(-) and lipid peroxidation. In addition, exogenous application of ACh improved plant water status by improving the accumulation of proline and regulating the stomatal opening and closing. Besides, ACh-induced amelioration of oxidative stress was related to the up-regulation of antioxidant enzyme activities like SOD, POD, CAT, and APX. Hence, it can be concluded that ACh treatment improved photosynthesis in tobacco by regulating the stomatal and non-stomatal factors and up-regulation of the antioxidant system.