In this paper, enantioselectivities of D-amino acid oxidase (DAAO) in ten ionic liquids were investigated in detail. Among the ionic liquids studied, 3-methylimidazolium formate ([MIM][COO]) was able to trigger enzyme activation with L-Ala as substrate. The enzyme activity of DAAO reached a maximum of 4.26 U mg⁻¹ min⁻¹ in the presence of 40% [MIM][COO], while no activity toward L-Ala was observed when DAAO was incubated with other ILs. Moreover, DAAO activities towards other L-amino acids such as L-Pro and L-Arg were also improved in [MIM][COO], which indicated that the enzymatic enantioselectivity of amino acids was lowered. This was further confirmed by capillary electrophoresis, circular dichrosim and fluorescence analysis. Molecular optimization supported that the protein–ionic liquid interactions modulated the structure of the enzyme, especially in [MIM][COO], leading to relaxation in the enantioselectivity of DAAO. This observation and exploration might open a new window for modulation enantioselectivity resolution in ionic liquids.