The invention discloses a preparation method of a tissue-adhering hemostatic antibacterial nano film. The preparation method comprises the steps as follows: 1) preparing a tissue-adhering hemostatic antibacterial matrix material; 2) dissolving the matrix material obtained in the step 1) in water, obtaining a nano thin film by a spin-coating method, drying and removing a solvent after curing with UV (ultraviolet) light, and obtaining the desired tissue-adhering hemostatic antibacterial nano film; and dissolving a water-soluble polymer in water, constructing a sacrificial layer on the nano filmby the spin-coating method, wherein the sacrificial layer is convenient for practical operation and easy to be removed by water washing in subsequent use. The tissue-adhering hemostatic antibacterialnano film prepared by adopting the method of the invention is high in biosecurity and convenient and comfortable to use, can firmly adhere to tissues with different morphologies, has a good hemostaticantibacterial effect, and does not produce bacterial resistance.