The invention discloses bis-fluoroquinolone oxadiazole urea derivatives containing N-methyl gatifloxacin as well as a preparation method and an application of the derivatives. The general chemical structural formula of the derivatives is represented as the formula I shown in the description; in the general formula, R is -CH2-CH3, cyclopropyl or -CH2-CH2F, L is -Cl, -F, 1-piperazinyl, substituted piperazine-1-yl or nitrogenous heterocyclic ring, X is -CH, -N, -CF or -COCH3, or R and X jointly constitute an oxazine ring or a thiazine ring. According to the bis-fluoroquinolone oxadiazole urea derivatives, organic splicing of a bis-fluoroquinolone skeleton, an oxadiazole heterocyclic ring and functional ureas is realized, hopping and stacking of different pharmacophores are realized, antitumoractivity and selectivity of fluoroquinolone are improved, toxic and side effects on normal cells are reduced, and the derivatives can be used as antitumor active substances to develop antitumor drugswith brand-new structure.