The invention discloses a three-layer nanopore thin film and a preparation method and application thereof. The preparation method comprises the following steps that a base plate is provided, two-layerSi3N4 / SiO2 nano thin films deposit on the surfaces of the two sides of the base body, a layer of Si3N4 thin film deposits on the top SiO2 thin film layer, the thin film on one side of the base boy isetched to form a base body releasing window, the base plate is etched so that the three-layer nano thin film can be obtained, reactive ion etching is adopted to etch the Si3N4 thin film on the releasing window of the base body to achieve thinning, a suspended three-layer nano thin film structure is obtained, and helium ion beams are used for carrying out etching so that a three-layer nano throughhole can be obtained. According to the preparation method for the three-layer nanopore thin film, an Si3N4 / SiO2 layer is formed through deposition, the Si3N4 top layer is formed through atom deposition or molecule vapor deposition, the thicknesses of all layers are uniform and controllable, contact faces between the layers are flat, the quality of the thin layer is stable, the preparation methodis easy in operation, it does not need to thin the deposition layer, the production efficiency is improved, and the production cost is saved.