Abstract: To enhance the bioavailability of phytosterols and investigate the role of soybean oil bodies in hydrophobic bioactive compound delivery systems, this study utilized whey protein isolate (WPI) and soybean oil bodies (SOBs) as interfacial particles to prepare whey protein isolate-soybean oil body emulsions (WOE) for phytosterol delivery. The effects of varying pH and ionic concentrations on the particle size, zeta potential, and rheological properties of WOE were investigated systematically, as well as the changes in microstructure, free fatty acid release rate, and phytosterol bioavailability of WOE during the simulated digestion process. The results demonstrate that WOE was an oil-in-water emulsion formed by adsorption of WPI and SOBs at the oil-water interface. At pH 4, the Zeta potential of WOE approaches 0, indicating that the isoelectric point of WOE was close to 4. WOE exhibited greater stability under alkaline conditions. The rheological results showed that WOE exhibited non-Newtonian fluid properties at different pH and NaCl conditions, and G' was larger than G” at frequencies from 0.1 to 100 Hz, and WOE had an elastic-dominated gel property. During the simulated digestion phase in vitro, the number of large lipid droplets in the WOE gradually decreased as the digestion time increased. During the simulated intestinal fluid digestion, trypsin degraded the fat within the emulsion droplets and released free fatty acids, and the fat droplet size of WOE gradually decreased from 16.85 μm to 7.25 μm, and the release of free fatty acids reached 75.45 μmol/mL in less than 90 min, and the phytosterol bioavailability consistently increased to 72.23%, which was higher than that of free fatty acid release (38.45 μmol/mL) and phytosterol bioavailability (48.08%) in the pure soybean oil group, indicating that WOE had good digestive properties and improved phytosterol bioavailability. This study provides a theoretical reference for hydrophobic bioactivities delivery systems, as well as a basis for the application of novel emulsion systems in food, dietary supplements and drug delivery.