Microplastics (MPs) have distributed in agricultural soil. However, the effects of MPs on the growth of tobacco remain unclear. In this study, a pot experiment was conducted to evaluate the effects of linear low-density polyethylene (LLDPE) MPs at four different concentrations (0 mg·kg-1 as control, 10 mg·kg-1, 100 mg·kg-1, and 1000 mg·kg-1) on soil enzyme activity, physiological characteristics, and tobacco growth. The results showed that compared with the control, the treatments of 100 and 1000 mg·kg-1 significantly inhibited the activities of soil catalase (S-CAT) and soil sucrase (S-SC). Compared with the control, the 1000 mg·kg-1 treatment significantly altered root morphology, inhibited tobacco growth, and water content, resulting in a significant decrease in chlorophyll a content, catalase (CAT) and superoxide dismutase (SOD) activities in tobacco leaves, thereby incited a significant increase in malondialdehyde (MDA) content and peroxidase (POD) activity. Interestingly, the 10 mg·kg-1 treatment stimulated the activity of soil urease (S-UE) and root biomass. Overall, this study highlights the significant impact of MPs on soil enzymes, oxidative damage to tobacco, and inhibition of tobacco growth and development. It emphasizes the environmental risks of MPs pollution in soil, particularly for commercial crops like tobacco, and provides insights for controlling MPs abundance in the environment. Further research is needed to investigate the underlying mechanisms of MPs’ effects on metabolism and genes in the soil-tobacco-microbial system.