The invention discloses a preparation method of high-purity humulus lupulus total flavones, wherein the preparation method includes following steps: (A) performing ethanol reflux to a raw material, humulus lupulus, to obtain an extract; (2) treating the extract through a polyamide column and passing the extract through a macroporous resin column, and performing elution to obtain a solution; (3) decoloring the solution through activated charcoal and spray-drying the solution to finally obtain the humulus lupulus total flavones being high in content and good in appearances. The preparation method is simple in steps, is less in usage of organic solvents during the process, is low in cost and is safe in operation and free of environment pollution. The final product is good in appearances and is high in yield and content. The preparation method is suitable for industrial production, fully utilizes two different ways of the humulus lupulus for brewing beer and making drugs, and provides new approaches of maximum utilization of the humulus lupulus.