The enzyme-assisted aqueous extraction process (EAEP) is an environmentally friendly strategy that simultaneously extracts oil and protein from several food matrices. The aim of this study was to investigate the effects of pH (6.5−9.5), temperature (45−55 °C), solids-to-liquid ratio (SLR) (1:12−1:8), and amount of enzyme (0.5−1.0%) on the extraction and separation of oil and protein from almond flour using a fractional factorial design. Oil and protein extraction yields from 61 to 75% and 64 to 79% were achieved, respectively. Experimental conditions resulting in higher extractability were subsequently replicated for validation of the observed effects. Oil and protein extraction yields of 75 and 72% were achieved under optimized extraction conditions (pH 9.0, 50 °C, 1:10 SLR, 0.5% (w/w) of enzyme, 60 min). Although the use of enzyme during the extraction did not lead to significant increase in extraction yields, it did impact the extracted protein functionality. The use of enzyme and alkaline pH (9.0) during the extraction resulted in the production of more soluble peptides at low pH (5.0), highlighting possible uses of the EAEP skim protein in food applications involving acidic pH. The implications of the use of enzyme during the extraction regarding the de-emulsification of the EAEP cream warrant further investigation.