The invention discloses an application of a wild licorice root extract in preparation of a freshwater fish anti-stress agent and a preparation method thereof. The wild licorice root extract serving asthe freshwater fish anti-stress agent can improve the ability of resisting the stress of breeding high density, copper sulfate and trichlorfon of freshwater fish, reduce the stress reaction, morbidity and mortality of the freshwater fish in a high-density breeding environment, reduce the breeding risk and increase the incomes of breeders.