Soybean protein hydrolysates (SPH), by-product of enzyme-assisted aqueous extraction (EAEP), were a promising source of natural nutraceutical. Nevertheless, the bitterness of SPH has limited their use as food ingredients or additives. The objectives of the present study were to use two-step enzyme-assisted aqueous extraction to produce SPH with reduced bitterness and high nutritional value (antioxidant activity and peptide nitrogen levels). In this study, the optimum conditions for SPH production were obtained using Protex 6 L (2 h) followed by Protease A 2SD (3 h) in two-step hydrolysis. The bitterness of SPH was positively correlated with the hydrophobic amino acids (Ala, Leu, Ile, Trp, Phe, Tyr) and hydrophobic peptide fractions. Additionally, the produced potential bitter peptides of SPH-6L were identified and predicted by LC-MS/MS and bioinformatics. IceLogo plot and heat map revealed that Protease A 2SD tends to cleave Leu and Arg residues from the N-terminal in bitter peptide to degrade bitter peptide. Thus, it could be concluded that the utilization of multi-enzyme hydrolysis, could be a promising process for the production of SPH with low bitter and potential antioxidant activity, which offers a promising approach for enhancing the usability and high-valued utilization of EAEP.