Astragalus membranaceus, the major components of which are saponins, flavonoids, and polysaccharides, has been established to have excellent pharmacological activity. After ginseng, it is the second most used medicinal plant. To examine the utility ofA. membranaceusas a sprout crop for plant factory cultivation, we sought to establish a functional substance control model by comparing the transcriptomes of sprouts grown in sterile, in vitro culture using LED light sources. Having sown the seeds ofA. membranaceus, these were exposed to white LED light (continuous spectrum), red LED light (632 nm, 1.58 μmol/m2/s), or blue LED light (465 nm, 1.44 μmol/m2/s) and grown for 6 weeks; after which, the samples were collected for transcriptome analysis. Scanning electron microscopy analysis of cell morphology in plants exposed to the three light sources revealed that leaf cell size was largest in those plants exposed to red light, where the thickest stem was observed in plants exposed to white light. The total number of genes inA. membranaceusspouts determined via de novo assembly was 45,667. Analysis of differentially expressed genes revealed that for the comparisons of blue LED vs. red LED, blue LED vs. white LED, and red LED vs. white LED, the numbers of upregulated genes were 132, 148, and 144, respectively. Binding, DNA integration, transport, phosphorylation, DNA biosynthetic process, membrane, and plant-type secondary cell wall biogenesis were the most enriched in the comparative analysis of blue LED vs. red LED, whereas Binding, RNA-templated DNA biosynthetic process, DNA metabolic process, and DNA integration were the most enriched in the comparative analysis of blue vs. white LED, and DNA integration and resolution of meiotic recombination intermediates were the most enrichment in the comparison between red LED vs. white LED. The GO term associated with flavonoid biosynthesis, implying the functionality ofA. membranaceus, was the flavonoid biosynthetic process, which was enriched in the white LED vs. red LED comparison. The findings of this study thus indicate that different LED light sources can differentially influence the transcriptome expression pattern ofA. membranaceussprouts, which can provide a basis for establishing a flavonoid biosynthesis regulation model and thus, the cultivation of high-functionalAstragalussprouts.