Endogenous β-glucosidase catalyzes the conversion of conjugated isoflavones into their corresponding aglycones. The present study evaluated how ultrasound parameters influenced the activity of endogenous β-glucosidase and isoflavone contents by using Box–Behnken design. Independent variables were temperature (X1 = 35, 45, and 55 °C), time (X2 = 5, 15, and 25 min), and ultrasound intensity (X3 = 6, 15, and 24 W.cm-2). Observed responses were β-glucosidase activity and daidzin, glycitin, genistin, malonyl-β-daidzin, malonyl-β-glycitin, daidzein, and genistein contents. Soybeans (50 g) in water (75 mL) were subjected to ultrasound and the samples were brought to 25 °C, which was followed by lyophilization, milling, and deffating for further analysis. β-glucosidase activity was evaluated using a spectrophotometric method and isoflavones were identified and quantified by ultra performance liquid chromatography. Mathematical models were validated, fitted predictive purposes, and showed coefficient of determination (R2) that ranged from 0.71 (β-glucosidase activity) to 0.97 (genistein). Additionally, the lack of fit was not significant (p > 0.05). Ultrasound and temperature positively affected the conversion of glucoside isoflavones into their aglycone forms. Regardless of the time of exposure, the highest conversion of daidzein (0.165 μmol.g-1) and genistein (0.141 μmol.g-1) was noted at 55 °C and 24 W.cm-2, which reflects an increase of 85% in the content of genistein. However, only the temperature positively affected the activity of endogenous β-glucosidase (maximum activity of 0.172 UA.g-1, at 45 °C). Therefore, both temperature and ultrasound intensity exposure increased aglycone isoflavone contents whereas the ultrasound temperature increased the activity of β-glucosidase. A myriad of health benefits has been attributed to soybean isoflavones and the aglycone form has been pointed as the best form for absorption. The present study supports the use of ultrasound to enhance the content of soybean isoflavones in the aglycone form and reports the best temperature and exposure intensity.