The change in the activity of glucose oxidase subjected to an asymmetrical alternating current (AC) electric field is investigated via horseradish peroxidase (HRP)-coupled bioassay. The effect of the amplitude, frequency and enzyme concentration have been shown to affect the enzyme activity towards glucose oxidation. The decrease in the enzyme activity is directly related to the change in pH and temperature of the GOx solution during AC electrolysis. The enzyme activity reduces with increasing amplitude, enzyme concentration and decreasing frequency. Results from UV-vis, FT-IR and UV CD spectroscopy showed that the AC treated GOx samples undergo structural modifications.