The invention discloses acteoside and a separating preparation method thereof, and relates to natural medicine separating. The acteoside is named as 2-(3',4'-dihydroxyphenyl)ethyl-O-alpha-L-pyran rhamanopyranosyl(1->3)-beta-D-(4-O-coffee acyl) glucopyranoside, and the molecular weight of the acteoside is 624.59. The preparation method comprises the steps that the dry overground part of macfadyena unguis-cati is smashed and then added in an ethanol solution for leaching, and a leaching solution is concentrated and dried, then dispersed and suspended with a methanol solution and extracted with petroleum ether; the degreased and decolored methanol solution is concentrated, diluted with water and then processed through macroporous adsorption resin, elution and impurity removal are performed with a low-concentration ethanol solution, and then elution is performed with high-concentration ethyl alcohol; eluant containing the acteoside is collected, concentrated and then processed through reverse-phase silica gel column chromatography, and gradient elution is performed with a methanol-water system; elution fractions containing the acteoside are combined, concentrated and then processed through LH-20 gel, elution is performed with a methanol-acetone system, the elution components containing the acteoside are concentrated and dried, and then the acteoside is obtained.