Based on aqueous two-phase system of polyethylene glycol (PEG)/salt, a simple pretreatment approach was developed for the extraction and determination of salidroside in Rhodiola. The influence factors on phase behavior of forming biphasic and partition behavior of the ATPS was investigated. Studied the factors such as polyethylene glycol (PEG) molecular weight and the mass fraction, the kinds of salt, the salt mass fraction, ionic strength, salidroside initial concentration influence on salidroside extraction rate. Determine the optimum extraction conditions with orthogonal experiment, when the aqueous two-phase system is 20%PEG1000, 20% (NH4)2SO4 and 1% KCl, the extracting effect is best; the extracting rate is 95.32%.Compared with the conventional liquid–liquid extraction or solid-phase extraction, this method was more environmentally, more cost effective.