The enzyme-assisted aqueous extraction process (EAEP) enables the fractionation of food matrices into oil-, protein-, and fiber-rich fractions that can be further converted into food, feed, and fuel, without the use of flammable solvent. Our research group investigated the effects of extraction conditions on oil and protein extractability, recovery of the extracted oil (i.e., cream de-emulsification), functional (solubility, emulsification, foaming properties) and biological (in vitro digestibility and allergenicity) properties of almond proteins. At pilot-scale, the use of protease to assist the extraction increased oil extractability from 62 to 67% and protein extractability from 65 to 76% compared with the aqueous extraction process (AEP). In addition, the cream fraction produced by the EAEP was easier to be de-emulsified compared with the AEP cream, regardless of the de-emulsification strategy employed. Under optimum conditions, enzymatic de-emulsification of AEP and EAEP creams led to oil recovery yields of 65.8 and 93.0%, while recoveries of 65.5 and 81.1% were achieved by chemical de-emulsification, respectively. More importantly, the EAEP significantly affected the functional and biological properties of the extracted protein. Higher protein solubility (30 vs 12%) and emulsification capacity (492 vs 402 g oil/ g protein) of the EAEP skim were observed at acidic pH (5.0), where almond protein solubility is hindered, compared with the AEP skim. Similarly, higher in vitro digestibility (88 vs. 80%) and reduced immunoreactivity (75% reduction) were observed for the EAEP skim. These results highlight the importance of processing optimization to improve extractability and recovery while aiming target functional and biological properties.