The invention discloses a 3,3'-methene-difluoroquinolone derivative of a chiral oxazine quinoline ring as well as a preparation method and application of the 3,3'-methene-difluoroquinolone derivative. The 3,3'-methene-difluoroquinolone derivative has a chemical general structural formula I shown in the specification, wherein R represents cyclopropyl or ethyl or fluoroethyl; R1 represents a hydrogen atom or methyl or ethyl; R2 represents a hydrogen atom or methyl; X represents a nitrogen atom or a hydrocarbon (CH) group or a fluoro-substituted carbon atom (F-C) or a methoxyl-substituted carbon atom (CH3O-C). The 3,3'-methene-difluoroquinolone derivative of the chiral oxazine quinoline ring, disclosed by the invention, can be used for realizing the superposition of a difluoroquinolone framework and alpha, beta-unsaturated ketone pharmacophores, so that the antitumor activity of a new compound is improved, the toxic and side effects on normal cells can be reduced, and the 3,3'-methene-difluoroquinolone derivative can be used as an antitumor active substance for developing an antitumor drug of a totally new structure.