A fluorescence biosensing strategy based on graphene oxide (GO) was reported for simple, rapid, sensitive, and selective DNA detection by T7 exonuclease assisted target recycling amplification. Due to the super fluorescence quenching efficiency of GO, the fluorescein amiditelabeled signal probe was firstly adsorbed onto the surface of GO and the fluorescence was quenched. Owing to its excellent selectivity for double-stranded DNA, T7 exonuclease was chosen as a signal-amplifying biocatalyst to improve the detection sensitivity. In the presence of target DNA, the signal probe could bind with target DNA and form a DNA duplex structure to trigger the digestion of the signal probe by T7 exonuclease, leading to the recycling of target DNA and the increasing of fluorescence intensity. Upon the recycling use of target DNA, this method achieved a high sensitivity towards target DNA with a detection limit of 0.3 pmol/L, which was lower than previously reported for GO-based DNA biosensors. Moreover, it does not require complex modifications of the molecular beacon and time-consuming thermal cycling procedures. Thus, the simple strategy provides a universal biosensing platform for DNA detection and it could find wide applications in DNA damage analysis and diagnostics.