In recent years, studies on the interactions between nanoplastics (NPs) and higher plants have attracted widespread attention. However, higher plants seem to respond differently to different doses of NPs. Currently, few comprehensive studies have shown the responses of tobacco plants ( Nicotiana tabacum L.) to different concentrations of NPs, especially their molecular mechanisms. In this study, we treated hydroponic tobacco seedlings with 0, 1, 10 or 50 mg/L polystyrene nanoplastics (PSNPs) to explore the potential underlying mechanisms via a combined physiological and transcriptomic analysis. Physiobiochemical results suggested that 1 mg/L PSNPs increased the biomass, chlorophyll content, photosynthesis and nutrient (nitrogen, phosphorus and potassium) content of tobacco seedlings, which promoted plant growth, whereas 10 and 50 mg/L PSNPs induced ROS accumulation, enhanced superoxide dismutase and peroxidase activities, decreased the biomass, chlorophyll content and photosynthesis, and interfered with the normal nutrient and hormone balance, which contributed to the inhibition of tobacco seedling growth. The transcriptomic results indicated that PSNPs modulated the expression of genes involved mainly in phenylpropanoid and flavonoid biosynthesis, starch and sucrose metabolism, photosynthesis and the tricarboxylic acid (TCA) cycle. Our study provides an in-depth comprehension of the response of tobacco to different concentrations of NPs from physiobiochemical and molecular perspectives.