Five factors (enzyme concentration, substrate concentration, pH, incubation temperature, and incubation time) were initially screened for the conversion of isoflavone glucosides to aglycones in soy germ flour. The incubation temperature/time most significantly affected aglycone yield; subsequently, a full 5 (35, 40, 45, 50, and 55 °C) × 6 (1, 2, 3, 4, 5, and 6 h) factorial design and response surface methodology were employed to attain an optimal incubation time/temperature condition. The optimum condition producing soy germ flour with a high concentration of daidzein, glycitein, and genistein was as follows: soy germ flour:deionized water (1:5, w/v), β-glucosidase at 1 unit/g of soy germ flour, pH 5, and incubation temperature/time of 45 °C/5 h. Under this optimal condition, most isoflavone glucosides were converted to aglycones with daidzein, glycitein, and genistein of ≥ 15.4, ≥ 6.16, and ≥ 4.147 μmol/g, respectively. In contrast, the control soy germ flour contained 13.82 μmol/g daidzin, 7.11 μmol/g glycitin, 4.40 μmol/g genistin, 1.56 μmol/g daidzein, 0.52 μmol/g glycitein, and 0.46 μmol/g genistein.