Pharmacological studies have shown that podophyllotoxin (PTOX) has anti‐tumor, antiviral and anti‐inflammatory effects. More and more derivatives of PTOX, such as VM‐26, NK‐611 and GL‐331, have gradually been synthesized and widely used in clinical practice. Sinopodophyllum hexandrum rhizome is rich in PTOX, which is much higher than other PTOX source plants. At present, research on S. hexandrum mainly focuses on artificial cultivation, chemical compositions, pharmaceutical value and the biosynthesis pathway of PTOX. However, the researches are relatively scattered and systematic review on PTOX is very limited. Therefore, this study performed a comprehensive investigation in terms of the plant origin source, content profile, and biological activities, in order to provide an integrated reference for in‐depth research and clinical application of PTOX in the chemistry and pharmacy fields, which can promote the innovative utilization of S. hexandrum resources and research and development of new anticancer drugs.