Glutathione S-transferase omega (GSTO) is a recently identified Glutathione S-transferase (GST), and it has several known functions and variable distribution patterns in many organisms. In Drosophila, GstO2 exists as two isoforms, GstO2A and GstO2B. Despite the high sequence homology between the two GstO2 isoforms, they have different physiological functions. In the present study, we characterized the structural and molecular properties of Drosophila melanogaster GstO2 isoforms. Homology modelling of GstO2s using I-TASSER servers for protein structure and function prediction revealed that the two GstO2s have different electropotential surface distributions and different shapes of the substrate-binding sites. The recombinant GstO2s have native molecular weights of ∼60 kDa. GstO2s have similar optimum conditions for enzymatic reactions at pH 8.0 and 40 °C. The kinetic parameters of the reduction of dehydroascorbate by these two GstO2s were determined. Collectively, our results provide structural insights into the different substrate profiles of the GstO2 isoforms.