The cutting seedlings of red pulp pitaya ‘Taiwan No. 6’ was used as experiment material, pretreated with 150mg/L ABA, and treated under cold stress (8℃/0℃，12 h/12 h) for 10 days. The effects of Reactive oxygen species (ROS) content, antioxidant enzyme activity, relative conductivity (REC), Malondialdehyde (MDA) content and AsA-GSH cycle related indicators were determined in order to study the regulatory effect of ABA on ROS metabolism of Pitaya seedlings, and to clarify the mechanism of ABA improving cold tolerance of Pitaya seedlings from the perspective of ROS metabolism. The results showed that: (1) Pitaya seedlings showed watery stains and wilted yellowing due to 10 days cold injury stress, and the index of chilling injury (CI) reached 0.52. Pretreatment with 150mg/L ABA can reduce the cold damage, the CI was reduced to 0.20. (2) Cold stress broke the balance of ROS metabolism. It significantly increased the O2- production rate and hydrogen peroxide (H2O2) content, and made membrane lipid peroxidation intensified, MDA and REC increased significantly in pitaya seedlings. Pretreatment with exogenous ABA can significantly reduced H2O2 content, reduce membrane lipid peroxidation, and significantly reduced MDA and REC. (3) The activities of SOD, POD, and CAT significantly increased in pitaya seedlings under cold stress. However, pretreatment with exogenous ABA further enhanced the activities of SOD, POD and CAT. (4) Cold stress caused the activities of APX, DHAR, GR and MDHAR significantly increased, and the contents of AsA, GSH, DHA and GSSG also increased in pitaya seedlings. However, the ratio of AsA/DHA and GSH/GSSG decreased, ROS scavenging system was damaged. Pretreatment with exogenous ABA can enhance the ability of pitaya seedlings to scavenge ROS under cold stress though by enhancing the activities of APX, GR and MDHAR, the content of antioxidants AsA and GSH, and the ratios of AsA/DHA and GSH/GSSG. In conclusion, exogenous ABA can effectively improve the cold tolerance of pitaya seedlings. It can enhance the scavenging ability of ROS and maintain the balance of ROS metabolism by increasing the activity of antioxidant enzymes and the metabolic activity of AsA-GSH cycle in pitaya seedlings, thereby reducing the degree of membrane lipid peroxidation and electrolyte leakage, and effectively alleviating the cold damage of pitaya seedlings.