A novel process for effective separation of phospholipids, triacylglycerol and cholesterol from fresh egg yolk has been developed and validated in this study. Ethanol was the only organic solvent used in the whole procedure. Following initial separation of protein and total lipids by ethanol, most of solidified triacylglycerol was removed from total lipids by low temperature treatment of ethanol extracts within 10 h. Then, β-cyclodextrin (β-CD) was used to remove cholesterol from the remaining ethanol extracts and recycling of β-CD was also studied to obtain cholesterol and reusable β-CD powder. The highest cholesterol removal rate of nearly 99 % was obtained at β-CD: cholesterol molar ratio of 5:1, water addition of 15 g/g β-CD and reacting temperature of 50°C. Ethanol in residual ethanol extracts was removed for obtaining phospholipids by rotary evaporation. The phospholipids produced in this procedure without cholesterol could be safety used as emulsifiers in food or cosmetic industry.