NOVELTY - An acetyl harpagide reference substance is prepared by (A) extracting raw material, (B) enriching by macroporous resin, (C) preparing liquid phase separation, and (D) recovering product. The step (A) comprises grinding decumbent bugle herb into coarse powder; and adding 8-1 times 40-60% ethanol, 3 times refluxing and extracting for 2 hours each time, combining extracting liquid, and cooling to room temperature. The step (C) comprises filtering condensed solution of step (B) by porous filter film, and separating by preparing liquid phase spectrum column.    USE - Method for preparing acetyl harpagide reference substance (claimed).    ADVANTAGE - The method has simple and rapid process low consumption of solvent, and high yield. It is environmental friendly.    DETAILED DESCRIPTION - Acetyl harpagide reference substance is prepared by (A) extracting raw material, (B) enriching by macroporous resin, (C) preparing liquid phase separation, and (D) recovering product. The step (A) comprises grinding decumbent bugle herb into coarse powder; and adding 8-1 times 40-60% ethanol, 3 times refluxing and extracting for 2 hours each time, combining extracting liquid, and cooling to room temperature. The step (B) comprises absorbing extracting liquid which is cooled in step (A) by activated macroporous absorbing resin column at 2 bed volume (BV), eluting impurities by 2-4 column times of water and 1-3 column times of ethanol of 40%; and eluting target material by 1-3 column times of ethanol solution of 60%, collecting eluting liquid, and condensing to 1/10 of the original volume. The step (C) comprises filtering condensed solution of step (B) by porous filter film, separating by effective preparing liquid phase spectrum column, monitoring in on-line manner, and collecting target compound spectrum peak preparing solution. The step (D) comprises decompressing solution of step (C), recovering methanol, enriching water solution by activated macroporous absorbing resin column at 2-5 BV, eluting by 1-3 column times of 95% ethanol, condensing the eluent to dry, and drying solid for 6-8 hours at 50-70 degrees C to obtain acetyl harpagide reference substance.