The invention provides an application of peroxy-ergosterol in preparation of a drug for preventing or treating influenza virus infection, and provides a method for separating peroxy-ergosterol from isatis roots simultaneously. The application of a small molecular compound peroxy-ergosterol in preparation of the drug for preventing or treating the influenza virus infection is provided by the invention, so that the effective drug for preventing and treating clinical influenza currently is provided. The inventor finds that peroxy-ergosterol can effectively inhibit influenza virus induced host cells from overexpressing a pattern recognition receptor RIG-I for pathogen recognition, so that effects of abnormal immune inflammation and mediated apoptosis transduced and mediated with pattern recognition receptor downstream signals are inhibited.