Anoectochilus roxburghii and Anoectochilus formasanus are the major species of genus Anoectochilus used in traditional Chinese medicine for their abundant content of flavonoids and some other medicinal constituents. In recent years, their wild resources are gradually exhausted due to over-collection and ecological deterioration. Artificial cultivation and tissue culture are employed to increase production. In this study, the open reading frame, promoter and genomic sequences of the chalcone synthase (CHS) gene were cloned from these two species according to their transcriptome information, and used for expression analysis in response to the induction of phenylalanine, ultraviolet light and NaCl, and its effect investigation on accumulation of flavonoids. The results showed that the expression of the CHS genes was upregulated in response to these inductions and resulted in increasing accumulation of total flavonoids. However, the increased flavonoids induced by phenylalanine and ultraviolet light were mainly allocated into the anthocyanidin branch of flavonoids biosynthesis. Not only did it improved the medicinal value, but might have inhibitory effect on plant growth because of the increased malondialdehyde accumulation. Under the induction of appropriate concentration of NaCl, the medicinal constituents of flavonoids were increased without inhibition to plant growth.