The invention discloses an active site composition of isatis roots, as well as a preparation method and the application thereof. The active site composition of isatis roots is prepared from a general phenylpropanoid part of isatis roots, a total alkaloid part, a total organic acid part and a total polysaccharide part. The active site composition obtained by the invention has high effective component contents and accurate active components. Proved by experimental results, the active site composition of isatis roots has obvious antiviral activity and favorable antibacterial and antipyretic activities. After being combined, the effective components reach favorable synergy, exert the antiviral, antibacterial and antipyretic activities at a plurality of targets spots through multiple paths, can be used for treating a viral disease with the bacterial inflammation of a febrile disease and have a wider clinical application range. Moreover, the invention optimizes and designs the extracting and refining process according to the physicochemical properties of the effective components, the active parts obtained through preparation has high effective component contents, and the preparation method has strong maneuverability and can realize industrialized mass production.