Low temperature is an important factor limiting the growth, development and yield of pitaya. 2,4-Epibrassinolide (EBR) can enhance the resistance of plants to various stresses such as low temperature. In order to improve the cold resistance of pitaya and alleviate the damage caused by low temperature, the cutting seedlings of red pulp pitaya 'Taiwan No. 6' was sprayed with 0.5 mg/L EBR, and treated under low temperature stress (4℃/0℃, 12 h/12 h) for 7 days. Reactive oxygen species (ROS) content, antioxidant enzyme activity, relative conductivity (REC), malondialdehyde (MDA) content, fatty acid composition and content were determined to clarify the regulatory effect of EBR on the cold tolerance of pitaya seedlings. Furthermore, the relationship between antioxidant system and fatty acid unsaturation of membrane lipids under low temperature stress was studied. Low temperature stress (4℃/0℃, 12 h/12 h) increased the content of reactive oxygen species (ROS), significantly decreased the content of unsaturated fatty acids. The fatty acids unsaturation (UFA/SFA) and double bond index (DBI) reduced by 32.1% and 32.4% respectively, and significantly increased the content of malondialdehyde (MDA) and relative conductivity (REC), resulting in decreased membrane stability and increased membrane permeability. The plants showed obvious watery stains and slight wilting, and the index of chilling injury reached 0.60. Under low temperature, exogenous EBR treatment increased the activities of superoxide dismutase, peroxidase and catalase, effectively removed the excessive accumulated ROS, significantly reduced the production rate of superoxide anion and the content of hydrogen peroxide, alleviated the oxidation reaction between ROS and unsaturated fatty acids in membrane lipids, and increased the relative content of unsaturated fatty acids in pitaya seedlings. The proportion of saturated fatty acids decreased significantly, UFA/SFA and DBI increased significantly, EBR maintained the stability of cell membrane, reduced REC and the content of membrane lipid peroxidation products MDA, greatly reduced watery stains of plants, and the index of chilling injury was reduced to 0.25. It is becouse EBR can activate the antioxidant enzyme system and effectively remove a large number of accumulated ROS under stress, leading to reduce the double-bond fracture of unsaturated fatty acids in membrane lipids, alleviate the degree of membrane lipid peroxidation, maintain the stability and function of membrane membrane. In conclusion, under low temperature stress, exogenous EBR can effectively improve the cold tolerance of pitaya seedlings. Its mechanism is by increasing the activity of antioxidant enzymes in pitaya seedlings, remove excess accumulated ROS, and significantly increase the unsaturation of cell membrane fatty acids, so as to reduce the degree of membrane lipid peroxidation and electrolyte leakage, maintain the stability and function of cell membranes.