Dendrobium nobile has a high ornamental value and is one of the important parental plants in the hybridization group of Dendrobium. To explore the physiological and molecular mechanisms of D. nobile flowers in response to high-temperature stress, this study combined water potential, cell membrane permeability monitoring, ethylene-related compound content determination, and transcriptome analysis. Treatment groups were subjected to 36 ℃ high-temperature stress for 5 h, 10 h and 24 h, with a normal temperature culture as the control. The physiological characteristics of D. nobile flowers under 36 ℃ and the transcriptome response to high temperature were studied. The results shoed that 36 ℃ high-temperature stress had almost no effect on the water status and cell membrane permeability of D. nobile flowers within 24h. The content of ethylene precursor (ACC) in the flowers significantly increased in all three high-temperature treatment groups compared to the control (Dno-CK). The number of differentially expressed genes between the 5 h high-temperature treatment and the control group (Dno-CK vs Dno-5h), the 10 h high-temperature treatment and the control (Dno-CK vs Dno-10h), and the 24 h high-temperature treatment and the control (Dno-CK vs Dno-24h) was 25 204, 25 528 and 26 878, respectively, with the number of genes up-and downregulated was approximately 1∶1. As the duration of high-temperature stress increased, the total number of differentially expressed genes in D. nobile flowers gradually rose. KEGG enrichment analysis showed that the differential genes of the three comparison groups were enriched to the monoterpene biosynthesis pathway, indicating that high temperature promoted the synthesis of volatile terpenoids in D. nobile. Functional enrichment analysis indicated that most of the differentially ex-pressed genes in response to high-temperature stress were enriched in pathways related to genetic information processing and metabolic pathways, with the highest number of differential genes in the nucleotide excision repair, starch and sucrose metabolism, and protein processing in the endoplasmic reticulum pathways. This suggests that high temperature stimulates D. nobile flowers to enhance various metabolic activities in response to heat stress. Screening of differentially expressed genes revealed that heat shock protein (HSP) genes in D. nobile flowers were significantly up-regulated in response to high-temperature stress. The HSP genes responding to high-temperature stress within 24 h in D. nobile flowers were primarily the HSP20 genes, followed by the HSP90 genes. Further research on the heat re-sponse genes of D. nobile flowers can focus on the HSP20 and HSP90 genes. This study on the physiological and transcriptome response of D. nobile flowers to high-temperature stress would provide a reference for heat-tolerant breeding research and theoretical support for the cultivation of heat-resistant D. varieties.