The purpose of this work was to prepare salidroside nano-liposomes by the ethanol injection method. To obtain the higher encapsulating efficiency of salidroside nano-liposomes, several factors including salidroside-loading capacity, cholesterol, Tween 80, ion strength and lipid concentration were investigated. The higher encapsulating efficiency of salidroside, 45%, was obtained with cholesterol to lipid mass ratio of 1:4, Tween 80 and lipid to the molar ratio of 1:2, and ion strength in a range 20–50 mmol/L. With the optimization operation, the particles of nano-liposomes were below 100 nm and zeta potential was in the range of −10 and −20 mV. The release study of salidroside in vitro from nano-liposomes exhibited a prolonged release profile as studied over a period of 24 h.