Abstract Panax notoginseng is a famous perennial herb widely used as materials of medicine and health-care food. Due to its various therapeutic effects, research work on P. notoginseng rapidly increased in recent years, urging a comprehensive review of the research progress of this important medicinal plant. Here, we summarized the latest studies on the representative bioactive constituents of P. notoginseng and their multiple pharmacological effects like cardiovascular protection, anti-tumor, and immunomodulatory activities. More importantly, we emphasized the biosynthesis and regulation of ginsenosides, which is the main bioactive ingredients of P. notoginseng. Key enzymes and transcription factors (TFs) involved in the biosynthesis of ginsenosides were reviewed, including diverse CYP450s, UGTs, bHLH, and ERF TFs. We also constructed a transcriptional regulatory network based on multi-omics data and predicted candidate transcription factors mediating the biosynthesis of ginsenosides. Finally, the current three major biotechnological approaches for ginsenoside production were highlighted. This review covered advances in the past decades, providing insights into quality evaluation and perspectives for the rational utilization and development of P. notoginseng resources. Modern omics technologies facilitate the exploration of the molecular mechanisms of ginsenoside biosynthesis, which is crucial to the breeding of novel P. notoginseng varieties. The identification of functional enzymes for biosynthesizing ginsenosides will lead to the formulation of potential strategies for the efficient and large-scale production of specific ginsenosides.