Tree peony (Paeonia suffruticosa Andr.) is a traditional Chinese flower, which prefers cool weather. However, high temperature in summer in the middle and lower reaches of the Yangtze River restricts its growth and development. In this study, osmotic regulation, antioxidant enzyme activities, and photosynthetic characteristics of tree peony in response to high-temperature stress were investigated. The results showed that high-temperature stress had destroyed the cell membrane, manifested as the increased relative electrical conductivity and malondialdehyde content. Moreover, high-temperature stress led to excessive accumulation of reactive oxygen species, thereby, activating antioxidant enzyme activities. Also, photosynthetic parameters and chlorophyll fluorescence parameters directly reflected the damage to the photosystem II reflection center under high-temperature stress. In addition, high-temperature stress led to stomatal closure and chloroplast damage. This study revealed the physiological responses of tree peony to high-temperature stress, laying a foundation for the promotion of tree peony in high-temperature areas and the improvement of high-temperature resistance.