Soy whey, a by-product of soy processing, has shown promise as a substrate for mycoprotein production using Aspergillus oryzae. However, the low biomass concentration obtained necessitates optimization of cultivation conditions to enhance total protein production. In this study, we optimized substrate concentration (50%, 75%, and 100%), initial pH (unadjusted, 4, 5, and 6), and supplementation with 8 g/L ammonium sulfate, minerals (0.75 g/L MgSO₄·7H₂O, 1 g/L CaCl₂·H₂O and 3.5 g/L KH₂PO₄), or their combination to maximize biomass production. The results showed that adjusting the initial pH to 5 and adding ammonium sulfate and minerals increased biomass concentration by 169% from 1.82 g/L to 4.9 g/L in 100% soy whey. This optimized condition also slightly improved the protein content of the biomass from 53% w/w to 55.93% w/w. Additionally, cultivating A. oryzae under these optimized conditions significantly reduced soy whey’s chemical oxygen demand from 8100 mg/L to 3267 mg/L, highlighting bioremediation potential. These findings suggest that the optimized conditions enhance the productivity of mycoprotein and also contribute to the sustainable management of soy whey waste, providing a combined benefit of nutrient recovery and wastewater treatment.