Abstract: By analyzing the physiological response of Phalaenopsis aphrodite leaves to CO₂ enrichment, and exploring the application of CO₂ fertilization in the greenhouse of Phalaenopsis aphrodite, it could provide theoretical and technical support for effectively improving the cultivation efficiency of Phalaenopsis aphrodite in greenhouse. The CO₂ enrichment experiment was carried out with the 2.5-inch cup seedlings of Phalaenopsis aphrodite Big Pepper as the materials, and the CO₂ releasing time (2 levels) and CO₂ concentration (4 levels) were taken as the investigation factors. A total of 8 treatments were set up according to the random block experiment. And the samples were taken on the 1 st, 3 rd, 8 th, 18 th and 30 th day after the treatment of CO₂ enrichment to analyze the effects of CO₂ enrichment on the content of osmotic adjustment substance and antioxidant enzyme activity in the leaves of Phalaenopsis aphrodite. The results showed that: Whether at AM 8:00-11:00 or PM 19:00-23:00, the MDA content, soluble protein content, total phenolic content, SOD activity, POD activity and CAT activity in the leaves of Phalaenopsis aphrodite showed a downward trend after 18 days when the the concentration of CO₂ was 400, 600 and 800 μmol/mol. However, when the concentration of CO₂ enrichment was 1 000 μmol·mol^-1, the content of soluble protein, POD activity and CAT activity in the leaves of Phalaenopsis aphrodite still showed an upward trend on the 30 th day. The results showed that the leaves of Phalaenopsis aphrodite began to adapt to the environmental conditions of CO₂ enrichment after 18 days when the concentration of CO₂ enrichment was below 800 μmol·mol^-1.