In order to optimize the Ultraviolet (UV) spectrophotometric method for determination of polyphenols inhibiting LOX activity, and to explore the influences of the buffer systems and pH on LOX enzyme activity and polyphenol auto-oxidation, this paper studied the changes of LOX enzyme activity and the auto-oxidation of gallic acid, quercetin, catechin and 3,4-dihydroxyphenylacetic acid under different buffer systems and pH conditions. The results showed that the activity of LOX was stronger under alkaline conditions, and the 4 polyphenols had the weakest self-oxidation in the borate system and the strongest self-oxidation in the Tris-HCl system. The higher the pH in each system, the stronger the self-oxidation of the 4 polyphenols, and the more intermediate and final products of auto-oxidation reflected in the UV spectrum. Finally, the pH 7.5 borate buffer was determined as the detection buffer system. It was found that LOX enzyme activity decreased first and then rised with the increase of 4 kinds of polyphenol concentration. The inhibitory effect of 4 kinds of polyphenols on LOX enzyme activity was in order: quercetin＞catechin＞gallic acid＞3,4-Dihydroxyphenylacetic acid.