To maximize value and minimize waste in soy processing, all major soybean components, i.e., oil, protein and carbohydrate, should be collected and utilized. Existing processing was originally designed to maximize oil extraction. It tends to make protein and carbohydrate separation from remaining meal more difficult and reduce their value. In this study, a single-step sonication-assisted enzyme processing was developed to separate and collect intact oil bodies, protein bodies and hydrolyzed carbohydrates from soybeans. Confocal laser scanning microscopic observations confirmed enzymatic destruction of cell wall and separation of oil bodies and protein bodies by centrifugation. Distributions of oil, protein and carbohydrate were quantitated in the top oil/cream layer, bottom protein-enriched precipitates, and the middle aqueous solution. Effects of the soybean particle size and the concentration of an Aspergillus niger enzyme were investigated. A pulsed ultrasonic treatment at 1.5 W/ml, for 5 min every 3 h during the enzyme processing, was found to significantly improve the performance and separation of oil bodies from protein. 87% of oil bodies were collected from cracked particles of 0.42-1.19 mm in size using, per g particles, 2 ml enzyme that contained 0.62 FPU/ml cellulase, 93 U/ml xylanase, 5.8 U/ml pectinase and 6.4 U/ml alpha-galactosidase.