The invention discloses a fluorescent enhancement nano thin film and a preparation method thereof. The preparation method includes the steps of: 1) pre-treating a substrate to enable the surface of the substrate to be charged, and then forming a single-layer or a multi-layer film, of which the surface has positive charges, on the substrate; and 2) spray-coating the surface of the single-layer or a multi-layer film with metal nano particles, removing physical adsorption, and blow-drying the material. The spray-coating pressure is 5-60 psi, the spraying time is 2-30 s, and the distance between a nozzle of a spray gun for spray coating and the substrate is 10-30 cm. The preparation method is simple, is short in time consumption, is free of limit on substrate size and is suitable for large-scale production. The nano particles are sprayed onto the pre-treated substrate through spray coating to form the thin film with appearance and distance of metal nano particles being adjustable.