Temperature affects the growth and accumulation of carbohydrate in plants. To study the response of Dendrobium nobile to cold stimulation, by using room temperature as the control, four low-temperature gradients (8 °C, 4 °C, 0 °C, −4 °C) were set to investigate the physiological changes and polysaccharide content of D. nobile. The results showed that under −4 °C treatment, the plant suffered severe damage and normal physiological activities were inhibited, resulting in inability to grow normally. Meanwhile, treatment at 0 °C for 4 d significantly increased the accumulation of polysaccharides in D. nobile (an increase of 56.18 % compared to CK), and the plant can grow normally after relieving cold stimulation. To explore the mechanism of its promoting polysaccharide accumulation, we conducted transcriptome analysis of D. nobile in this treatment. The results showed that TPP6, DPE1, PGMP, BAM3 genes in the starch and subcrosse metropolis; The pel gene in pentose and glucose conversions and transcription factors such as bHLH, MYB, and WRKY were significantly upregulated, thereby promoting the accumulation of polysaccharides in D. nobile. This study provides new perspective into the low-temperature adaptability of D. nobile and lays the foundation for further research on the biosynthetic pathways of D. nobile polysaccharides.