The invention discloses a fluorescence enhanced substrate which comprises a thin nano silver film with the thickness of 0.5 [mu]m to 1 [mu]m, wherein circular nano silver bulge arrays with the interval of 5 nanometers to 15 nanometers are arranged on the surface of the thin film; the diameter of the circular bulges is 90 nanometers to 218 nanometers; and the height of the circular bulges is 60 nanometers to 120 nanometers. The invention further discloses a preparation method of the fluorescence enhanced substrate. The preparation method comprises the steps of firstly, generating a titanium oxide nanotube on the surface of a titanium sheet through utilizing an anodic oxidation method; peeling the nanotube on the surface of the titanium sheet through utilizing an adhesive tape to leave meshy nanopores; sputteringnano silver into the meshy nanopores; and finally, taking down the thin nano silver film from the titanium sheet through utilizing a conductive adhesive tape, so as to enable the nano silver to be overturned upwards to form the circular bulges and to obtain the fluorescence enhanced substrate. Through the preparation method, the preparation technology of the fluorescence enhanced substrate is simplified, the fluorescence enhanced substrate with low cost is prepared and obtained, and a wide application prospect is achieved in the aspects of organic matter detection and optical sensors.