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Background: Spirodela polyrrhiza, a simple aquatic floating plant with great potential in synthetic biology. It has been noted that nitric oxide (NO) stimulates plant development and raises the biomass and flavonoid content in some plants. However, the molecular explanation on the mechanism of NO action is still unclear. Results: S. polyrrhiza was treated with various concentrations of sodium nitroprusside (SNP) as an NO donor. Physiological and transcriptomic analysis were performed in our study. The results showed that under low concentration SNP conditions, S. polyrrhiza alleviated malondialdehyde accumulation, increased levels of fresh weight, dry weight, starch, soluble protein, and flavonoids, and enhanced antioxidant enzyme activity. 2776 genes were found to have differential expression in the S. polyrrhiza treated with 0.025 mM SNP and control groups by RNA-Seq. Of these DEGs, in comparison to the controls, 1425 genes were up-regulated and 1351 genes were down-regulated. The findings of the qRT-PCR study revealed that the expression of genes involved in flavonoid biosynthesis, NO biosynthesis, as well as transcription factor (TF) was increased, while the expression of photosynthesis related-genes was decreased. Under SNP treatment, S. Polyrrhiza redirects metabolic flux of fixed CO2 into starch synthesis branch and flavonoid biosynthesis branches. Conclusions: The results provide new insights into the mechanisms causing the accumulation of starch and flavonoids by SNP treatment, meanwhile, The SNP-regulated genes would make excellent candidates for synthetic biology to increase the flavonoid content in S. Polyrrhiza.