The efficient, safe, and rapid application of pulsed electric field (PEF) technology to enzymatic modification was studied. The effect of PEF treatment on the enzymatic activity, properties, and kinetic parameters of glucoamylase were investigated, while conformational transitions were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD) and fluorescence spectroscopy. The results showed that the maximum increase in activity obtained was 20.74 ± 0.71% as compared to control, observed when 15 kV/cm electric field intensity and 80 mL/min flow rate were applied and this activation effect lasted for up to 17 h at 4 ℃. Moreover, PEF treatment widened the range of optimum temperature for glucoamylase activity, increased the Vmax and decreased the Km, but had a negligible effect on the optimum pH of glucoamylase. Results of SDS-PAGE indicated that the primary structure of glucoamylase did not change after PEF treatment. However, according to CD and ?uorescence spectra, PEF treatment enhanced the α-helix and β-sheet content in glucoamylase by 3.00% and 2.80%, respectively, leading to more ordered conformation. Additionally, intrinsic fluorescence spectra analysis showed that PEF treatment resulted in a red shift of the emission maximum of glucoamylase due to the exposed microenvironment of Trp residue. Therefore, based on these results, both, secondary and tertiary structures of glucoamylase were modified.