The effect of pulsed electric field (PEF) on the secondary and tertiary structure of lipoxygenase (LOX) in a buffer solution was analyzed using far UV-circular dichroism (CD) and fluorescence spectrophotometry, respectively. The secondary and tertiary structure of LOX changed after PEF treatment. The CD spectra of LOX also changed, with the intensity of two negative peaks and the content of alpha-helix significantly decreased (p < 0.05). The decrease in alpha-helix content in LOX showed a good linear correlation with the electric field strength. The fluorescence intensity of LOX increased, and the relative fluorescence intensity of the two characteristic peaks of LOX emission spectra at 337 nm and 583 nm also showed a good linear correlation with the electric field strength. These results showed that the activity inactivation of LOX may be due to the alteration in secondary structure, and both had a good relation.