Red light (RL) accelerated starch accumulation in S. polyrhiza, but higher protein content under blue light (BL) was associated with the upregulation of most DEGs enriched for specific GO terms and KEGG pathways. Red light (RL) and blue light (BL) greatly influence the growth and physiological processes of duckweed. Physiological and molecular mechanisms underlying the response of duckweed to different light qualities remain unclear. This study employed physiological and transcriptomic analyses on duckweed, Spirodela polyrhiza "5510", to elucidate its differential response mechanisms under RL, BL, and white light conditions. Changes in growth indicators, ultrastructure alterations, metabolite accumulations, and differentially expressed genes (DEGs) were measured. The results showed that BL promoted both biomass and protein accumulations, while RL promoted starch accumulation. A total of 633, 518, and 985 DEGs were found in white-vs-red, white-vs-blue, and red-vs-blue comparison groups, respectively. In Gene Ontology (GO) enrichment analysis, the DEGs in all three comparison groups were significantly enriched in two GO terms, carboxylic acid metabolic process and lyase activity. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the DEGs were greatly enriched in two pathways, histidine metabolism and isoquinoline alkaloid biosynthesis. Higher protein content under BL was associated with the upregulation of most DEGs enriched with the GO terms and KEGG pathways. Furthermore, the light qualities influenced the gene expression patterns of other metabolic pathways, like carotenoid biosynthesis, and the regulation of these genes may explain the level of photosynthetic pigment content. The results revealed the physiological changes and transcriptome-level responses of duckweed to three light qualities, thereby providing bases for further research studies on the ability of duckweed as a biomass energy source.