Thraustochytrids biomass, rich in docosahexaenoic acid (DHA), could be a sustainable ingredient for the aquafeed industry. However, its production cost renders it less competitive than fishmeal and fish oil from wild-caught fish. This study proposes optimizing the hydrolysis conditions of thraustochytrid biomass to generate an ingredient with improved properties thanks to the production of peptides with different biological activities. The improved nutritional value of the biomass could justify its use to decrease the amount of wild-caught fish in aquafeed, which would enhance the sustainability of fish aquaculture. First, two commercial proteases, Papain and Alcalase 2.4L, were compared for their capacity to hydrolyze the biomass. The best degree of hydrolysis (19 ± 1%) was obtained with Alcalase 2.4L, which was then used for the optimization of the pH, enzyme concentration and reaction time using response surface methodology. The results showed that the highest concentration of peptides and DHA in the aqueous phase was obtained with a pH, enzyme concentration and reaction time of 7.5, 2.7% and 205 min, respectively. If thraustochytrid hydrolysates prove to improve fish fitness, this mild and free-solvent process protocol could be used to produce a sustainable ingredient for aquafeed.