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Dendrobium nestor is a famous orchid species in the Orchidaceae family. There is a diversity of flower colorations in the Dendrobium species, but knowledge of the genes involved and molecular mechanism underlying the flower color formation in D. nestor is less studied. Therefore, we performed transcriptome profiling using Illumina sequencing to facilitate thorough studies of the purple color formation in petal samples collected at three developmental stages, namely—flower bud stage (F), half bloom stage (H), and full bloom stage (B) in D. nestor. In addition, we identified key genes and their biosynthetic pathways as well as the transcription factors (TFs) associated with purple flower color formation. We found that the phenylpropanoid–flavonoid–anthocyanin biosynthesis genes such as phenylalanine ammonia lyase, chalcone synthase, anthocyanidin synthase, and UDP-flavonoid glucosyl transferase, were largely up-regulated in the H and B samples as compared to the F samples. This upregulation might partly account for the accumulation of anthocyanins, which confer the purple coloration in these samples. We further identified several differentially expressed genes related to phytohormones such as auxin, ethylene, cytokinins, salicylic acid, brassinosteroid, and abscisic acid, as well as TFs such as MYB and bHLH, which might play important roles in color formation in D. nestor flower. Sturdy upregulation of anthocyanin biosynthetic structural genes might be a potential regulatory mechanism in purple color formation in D. nestor flowers. Several TFs were predicted to regulate the anthocyanin genes through a K-mean clustering analysis. Our study provides valuable resource for future studies to expand our understanding of flower color development mechanisms in D. nestor.