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<i>Siraitia siamensis</i> is a traditional Chinese medicinal herb. In this study, using <i>S. siamensis</i> cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of <i>cucurbitadienol synthase</i>, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that <i>CDC6</i> and <i>NCBP2</i> expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in <i>S. siamensis</i> in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways.