In this work, different antinutritional factors (trypsin inhibitors, isoflavones and raffinose family oligosaccharides) were selectively removed from soy flour by using aqueous micellar two-phase systems (AMTPS). The effects of independent variables including temperature (30–60 °C), time (10–40 min) and solid to liquid ratio (0.025–0.050 g/L) on the extraction of each antinutritional factor were analyzed using a full factorial design. As general tendency, temperature and time were the most significant parameters (p < 0.05). The best condition for the selective recovery (97% of isoflavones at top phase, and more than 50% of the rest of ANFs at bottom phase) were 5 g/L of Genapol X-080, 0.2 moL/L of sodium citrate pH 5.00, 30 °C, 40 min and 0.050 g/L. Besides, in vitro gastrointestinal digestions assays demonstrated that the treated soy flour improved its protein digestibility. The findings of this work represent the introduction of a novel methodology to selectively remove soy antinutritional factors.