Panax ginseng C. A. Mey. is an important plant species used in traditional Chinese medicine, whose primary active ingredient is a ginsenoside. Ginsenoside biosynthesis is not only regulated by transcription factors but also controlled by a variety of structural genes. Nonetheless, the molecular mechanism underlying ginsenoside biosynthesis has always been a topic in the discussion of ginseng secondary metabolites. Squalene epoxidase (SQE) is a key enzyme in the mevalonic acid pathway, which affects the biosynthesis of secondary metabolites such as terpenoid. Using ginseng transcriptome, expression, and ginsenoside content databases, this study employed bioinformatic methods to systematically analyze the genes encoding SQE in ginseng. We first selected six PgSQE candidates that were closely involved in ginsenoside biosynthesis and then identified PgSQE08-01 to be highly associated with ginsenoside biosynthesis. Next, we constructed the overexpression vector pCAMBIA3301-PgSQE08-01 and the RNAi vector pART27-PgSQE08-01 and transformed ginseng adventitious roots using Agrobacterium rhizogenes, to obtain positive hairy-root clones. Thereafter, quantitative reverse transcription-polymerase chain reaction and high-performance liquid chromatography were used to determine the expression of relevant genes and ginsenoside content, respectively. Then, we focused on the function of PgSQE08-01 gene, which was noted to be involved in ginsenoside biosynthesis. Thus, these findings not only provided a molecular basis for the identification of important functional genes in ginseng but also enriched genetic resources for the biosynthesis of ginsenosides using synthetic biology.