Abstract: Effects of different mobile phases, flow rates, dectecting warelengths and temperatures on cholesterol separation of dried duck egg yolk were studied by direct saponification-RPHPLC. The optimal chromatographic conditions of RP-HPLC are as follows: mobile phase is acetonitrile∶methol∶isopropanol=50∶80∶10(v/v/v); UV detecting wavelength is 205 nm; flow rate is 1.3 mL/min; column temperature is 45℃. Cholesterol content of the dried natural red yolk of duck egg and the common dried duck eggs yolk are 24.84 mg/g and 25.75 mg/g respectively under the above conditions.