Growth temperature for tobacco plants greatly influences growth and quality of tobacco leaves, and polyphenol content in tobacco leaves was often related to tobacco aroma flavour types. The purpose of this study was to evaluate the effect of different growth temperatures (mean temperature of 30.5, 23.5, and 16.5 °C) on polyphenol content, key enzyme activities and gene expression of polyphenol metabolic pathways in tobacco leaves during the growth and development process of tobacco plants grown in artificial climate chambers. Compared to the leaves of plants grown at a mean temperature of 23.5 °C, tobacco leaves grown in a lower temperature environment (16.5 °C) had higher chlorogenic acid, neochlorogenic acid, rutin, scopoletin, resurrection lily phenolic glycosides, caffeic acid, total phenol, total flavonoids, and lignin content. Plants grown in a higher temperature environment (30.5 °C) had lower chlorogenic acid, neochlorogenic acid, scopoletin, resurrection lily phenolic glycosides, caffeic acid, total phenol, total flavonoids, and lignin content than those grown at 23.5 °C. The activities of PAL and C4H enzymes, key enzymes in polyphenol metabolic pathways, were higher in plants grown at a low temperature (16.5 °C) than in plants grown at higher temperatures. In contrast, PPO and POD enzyme activities were lower in plants grown at low temperatures than in plants grown at higher temperatures. These results indicate that growth temperatures can significantly affect the polyphenol content of tobacco leaves and that lower growth temperatures are advantageous for the synthesis and accumulation of polyphenols.