The invention discloses a fleroxacin aldehyde thiosemicarbazone derivative and a preparation method and application thereof. A chemical structural formula adopts a formula I which is shown in the description, wherein the substituted group R is H (hydrogen) atom or hydrocarbyl or cyclopropyl with 1 to 5 carbon atoms. The fleroxacin aldehyde thiosemicarbazone derivative has the advantage that the three advantage pharmacophores of fleroxacin keel, Schiff base imine and thiourea are spliced, so that the anti-tumor activity of a new compound is increased, the toxic or side effect to normal cells is decreased, and the fleroxacin aldehyde thiosemicarbazone derivative can be used as an anti-tumor activity matter to develop an anti-tumor medicine with a fully new structure.