The substituent effect of intramolecular proton transfer of N- (2-hydroxybenzylidene) aniline derivatives were studied at the B3LYP/6-31+ G (d, p) level. The results show that the introduction of the electron withdrawing group make the molecules more tending to the planar configuration, and the electron donor replace the molecules to a certain degree of torsion. After the introduction of the electron-withdrawing substituents, the electron density of the molecular plane is reduced The N1-H2distance enhances the intramolecular hydrogen bondstrength and reduces the structure of the alcohol to ketone. The electron recombination of the electron donor increases the electron density of the molecular plane, increases the N1-H2 spacing, reduces the intensity of the intramolecular hydrogen bonds, and increases the proton transfer energy barrier. The study of the molecular prospective orbit shows that the alcohol is more likely to form an excited state than the ketone structure, and the energy gap decreases with the increase of the electron-withdrawing ability and the electron-supplying ability, which indicates that the biological activity becomes stronger.