The invention provides a characteristic chromatogram establishing method of a traditional Chinese medicine or a traditional Chinese medicine composition preparation, and application. The characteristic chromatogram establishing method comprises the following steps of: extracting the traditional Chinese medicine or the traditional Chinese medicine composition preparation by using a solvent to prepare a test solution; and carrying out high performance liquid chromatography determination on the test solution, and establishing a characteristic chromatogram, wherein acetonitrile(A)-acid aqueous solution(B) is used as a mobile phase, gradient elution is adopted, and the detection wavelength is 190-500 nm. The characteristic chromatogram establishing method of the traditional Chinese medicine ortraditional Chinese medicine composition preparation is clear in characteristic peak attribution and free of negative interference, and can be used for establishing the characteristic chromatogram ofthe traditional Chinese medicine or traditional Chinese medicine composition preparation containing natural calculus bovis, artificial calculus bovis, natural musk, artificial musk, scutellaria baicalensis, coptis chinensis, cape jasmine fruits and radix curcumae.