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Denphal-type Dendrobium is the famous cut and potted flower in the world, and most cultivated in tropical and subtropical regions. However, it often suffers from cold in winter in subtropical regions. To verify the physiological response of Denphal-type Dendrobium under low temperature in this study, the mature and young plantlets of Dendrobium Udomsri Beauty were treated under 15, 10, and 5 °C, respectively. And then the electrical conductivity (EC), soluble protein, soluble sugar, free proline, malondialdehyde (MDA), chlorophyll content, and the rate of defoliation after regrowth were measured. The results showed that both mature plant and young seedlings of Dendrobium Udomsri Beauty, the EC, soluble protein, soluble sugar, free proline, MDA content, and defoliation rate were increased with the decrease of treatment temperature and the extension of treatment times. The content of chlorophyll decreased gradually with the decrease of treatment temperature and the extension of treatment times. The correlation analysis showed that soluble sugar, free proline, MDA, chlorophyll content, and defoliation rate were significantly correlated with the semi-lethal temperature. It is indicated that the content of free proline, MDA, chlorophyll, and defoliation rate could be used as the effective indexes for the comprehensive assessment of cold tolerance of Dendrobium Udomsri Beauty.