Paeonia ostii is an economically important oil crop, which has been widely cultivated in the middle and lower reaches of the Yangtze River in China in recent years. Although P. ostii is highly adaptable to the environment, the prolonged high summer temperature in this region severely inhibits its growth, which adversely affects seed yield and quality. In this study, P. ostii plants were subjected to 20°C/15°C (day/night) and 40°C/35°C (day/night) temperatures for 15 days. The changes in physiological and biochemical indicators of P. ostii under high-temperature stress were initially investigated. The results showed that with the deepening of leaf etiolation, chlorophyll a and chlorophyll b concentration, carotenoid concentration, Soil Plant Analysis Development (SPAD) values and leaf relative water content decreased significantly, while both relative electrical conductivity (REC) and free proline concentration showed an upward trend. Meanwhile, the continuous accumulation of reactive oxygen species (ROS) in P. ostii plants, led to an increased activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX). Moreover, with the extension of the high-temperature treatment, the anatomical structures of P. ostii were destroyed, resulting in a decreased photochemical efficiency of the photosystem II (PSII) reaction center and photosynthesis was inhibited. Taken together, these results provide reference values for understanding the physiological response of P. ostii to high-temperature stress and establish a foundation for further research on the relevant underlying molecular mechanisms.