Egg yolk is a rich source of bioactive compounds such as immunoglobulin Y, phospholipids, carotenoids, and amino acids. Supercritical carbon dioxide (SC-CO2) has been used a green solvent for lipid extraction from egg yolk to a limited extent due to low extraction yield. Cholesterol and phospholipids have been used extensively in pharmaceutical and cosmetic applications. The objectives of this thesis research were to investigate the effects of enzymatic hydrolysis and pellet preparation on the extractability of lipids (neutral lipids, cholesterol and phospholipids) using a two-step SC-CO2 extraction process and to characterize the fractions obtained. In the first study, egg yolk was treated with a combination of Protease M and Lipase AY-30, which facilitated the formation of a cream layer and a liquid subnatant fraction. Pretreatment of egg yolk could be a viable approach to facilitate destruction of the lipoprotein assembly and to release the lipids. The cream had a higher lipid content (82%) compared to egg yolk (63%). Lipids were extracted in two steps using SC-CO2 at 48.3 MPa, 70 °C and CO2 flow rate of 1 L/min (at ambient conditions). In the first step, neutral lipids were extracted using neat CO2 in which most of the cholesterol was recovered. Polar lipids were extracted in the second step with the addition of 8% (mole%) ethanol as co-solvent. Total extract yield (neutral lipids, cholesterol and phospholipids) from cream was higher than that from egg yolk. The recoveries of cholesterol and PL were 98% and 93%, respectively, based on the feed material used for extraction. Microstructure analysis results showed dissociated individual particles (granules) in the residue samples after SC-CO2 extraction. The second study focused on the characterization of the potential biological activity such as antioxidant activity of the residue sample after SC-CO2 extraction. The subnatant phase after hydrolysis and the residue cream sample after SC-CO2 extraction were rich in small molecular weight peptides. Cellular superoxide generation was detected by dihydroethidium (DHE) to determine the antioxidant activity of egg yolk proteins of the cream residue and the subnatant. The subnatant showed an antioxidant effect in vascular smooth muscle cells (VSMCs) and endothelial cells against tumor necrosis factor-α (TNF-α) by suppression of the reactive oxygen species (ROS) mediated fluorescence. The findings demonstrate the potential for new opportunities for value-added applications of egg yolk fractions.