The invention relates to the field of separation of natural compounds, in particular to a novel compound in herba hedyotidis diffusae, and application of the novel compound. The invention discloses the compound diffusoside C separated from an aqueous extract of the herba hedyotidis diffusae, and provides the application of the compound diffusoside C in preparation of medicines for resisting EV71 virus. Systematic separation is performed on 30% of an ethanol elution site of the aqueous extract of herba hedyotidis diffusae, and separation techniques such as silica gel column chromatography, MCIgel column, Sephadex LH-20 gel column chromatography, ODS medium pressure liquid chromatography and reverse phase semi-preparative high performance liquid chromatography (HPLC) are comprehensively used; the novel compound diffusoside C is obtained in the separation process; furthermore, the compound diffusoside C is found to have good anti-EV71 virus activity, and be in therapeutic window and safeto use in a research process; therefore, the novel compound has an important research significance when being applied to preparation of the medicines for resisting the EV71 virus.