The invention discloses a preparation method of a zinc oxide-graphene oxide composite nanomaterial, which is implemented through the following steps: by taking soluble divalent zinc ions as a zinc source, taking the soluble divalent zinc ions, graphene oxide and alkali liquor as reactants together, according to a situation that the mass ratio of the zinc ions to the graphene oxide is (1-3): 1, forming a solute composition; by taking alcohol as a solvent, forming a reaction solution mixed system; and after a reaction is performed for 30 min-5 h, purifying a substrate by using centrifugation and washing methods, so that the zinc oxide-graphene oxide composite nanomaterial is obtained. The zinc oxide-graphene oxide composite nanomaterial prepared according to the invention has an effective antibacterial ability at low concentration, and has no obvious toxic action on cells. According to the invention, the operation process is simple, the reaction process has no obvious pollution to the environment, the production cost is low, and the prepared nanomaterial can be applied to the antisepsis of medical apparatuses and instruments, and has a potential in commercial large-scale production.