Soy isoflavones have been widely investigated due to their health-enhancing properties. However, to be efficiently absorbed, they must be transformed from glycosylated to aglycone form. Moreover, studies have shown that the clinical efficacy of isoflavones is related to the capacity of producing equol, a daidzein metabolite formed by intestinal bacteria. Thereby, the enzymatic biotransformation of soymilk by tannase was a process studied by our research group with demonstrated potential. This study aimed to investigate the optimization parameters of soymilk biotransformation process by tannase, in order to generate a commercial source of aglycones and equol. The traditional enzymatic biotransformation of soymilk with β-glycosidase was performed as control for the new proposed process. The enzymatic optimization process was followed by HPLC-DAD and four variables were tested: hydrogen potential, temperature, enzyme concentration and reaction time. The results showed that the best conditions which increased the bioactive isoflavones content were: 10% of enzyme concentration, pH 6.5, temperature of 50°C and reaction time of 45 min. The analysis demonstrated that the aglycones concentration, after the reactions with tannase, has increased in 47.8-53.8 and 54.2-63.6 times to daidzein and genistein, respectively, compared to untreated soymilk. These results suggest that the glycosidic forms of isoflavones were converted into aglycones, as well other substrates were also transformed into daidzein and genistein. Moreover, the equol metabolite was identified in “soymilk + free tannase”, corresponding to 0.93% of all compounds present in this sample. This finding is probably due to the higher performance of free form of tannase, acting specifically on daidzein metabolism. In conclusion it can be said that the enzymatic biotransformation is an efficient strategy to improve the bioactive potential of soymilk. The use of tannase proved that it is possible to produce equol using only enzymatic bioprocess, without microbial intervention, confirming the great potential of this enzyme.