Dendrobium nobile Lindl (D. nobile) is a traditional Chinese medicinal plant that has high medicinal value. Different duration of red and blue light plays vital roles in the regulation of growth and main medicinal ingredients in plants. Based on the optimal red-blue combination of light (R: B=1:2) screened in the previous stage, we used RNA sequencing technology to explore the effects of irradiation duration of 3 h (A), 6 h (B), 9 h (C), and 12 h (D) on the growth and main medicinal components. The results showed that different lengths of the combined red and blue light could significantly promote photosynthesis and antioxidant system, and increase main medicinal ingredients content in D. nobile within 120 d. Besides, group B had the most obvious effect on promoting growth at 30–60 d. The accumulation of polysaccharides, flavonoids, and other major medicinal components was maximum within 30–60 d. Moreover, the transcriptomic analysis showed that there were 4646 differential genes between group B and CK, of which 1952 were up-regulated, and 2694 were down-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses revealed that AUX1, AUX/IAA, GH3, SAUR, DELLA, TF, ARR-A, ARR-B, TCH4 in the phytohormone signaling pathway, PAL, COMT, POD, β-Glucosidase in the phenol propane biosynthesis pathway, ISA, OTSB, GN4 and other genes in the starch and sucrose metabolic pathway are key genes in group B which regulate the physiology and main medicinal ingredients of D. nobile. In conclusion, this study provides a preliminary interpretation of the effects of different irradiation durations of red and blue combined light on the growth and accumulation of medicinal components of D. nobile, and provides some theoretical and experimental basis for its high-quality artificial cultivation.