The invention discloses a fingerprint spectrum of isodon lanceolata and an establishment method and application thereof, the method comprises preparation of a test sample and high performance liquid chromatography detection conditions, and the high performance liquid chromatography detection conditions are as follows: a chromatographic column is a Kromasil 100-5C18 chromatographic column (250mm*4.6mm, 5[mu]m); a mobile phase is a methanol -0.1% glacial acetic acid aqueous solution; a gradient elution mode is adopted: 0 min, 10 min, 26 min and 60 min, and methanol is 20%, 35%, 37% and 70%; the sample size is 3-10 [mu]L; and the detection wavelength is 239 nm, the flow velocity is 1 mL / min, and the column temperature is 20-35 DEG C. According to the isodon lanceolatus medicinal material fingerprint spectrum established by the invention, 12 common peaks are determined, through the abundant chromatographic peak information and the evaluation of the similarity of the common peaks, the overall evaluation of the quality of the medicinal material is facilitated, the uniformity and stability of the internal quality of the medicinal material are ensured, the quality of the isodon lanceolatus medicinal material can be effectively monitored, and the authenticity of the isodon lanceolatus medicinal material can be effectively identified.