Protein antigenicity can be reduced by enzymatic hydrolysis, which can be performed either by free or immobilized enzyme. The immobilized enzyme is removed from the reaction medium and reused, while the free enzyme must be inactivated to stop the reaction, generally by heating. Here we have shown that hydrolysates produced with free or immobilized Alcalase on glyoxyl-agarose bead presented different physicochemical properties (hydrophilicity profile, molecular mass distribution, surface hydrophobicity) and different levels of residual milk allergens (α-lactalbumin and β-lactoglobulin). Although, under the studied conditions, the hydrolysis with immobilized enzyme did not reduce the residual allergen levels as efficiently as the free enzyme, the results suggest potential applications of immobilized Alcalase for production of hypoallergenic hydrolysates.